The 125-year-old Dundee-based business said it was taking the precaution in order to protect its customers regardless of the outcome in September.
The trust currently has offices in Dundee, Edinburgh and London.
In a statement outlining the company's annual results, chief executive Katherine Garrett-Cox said: "2014 is an important year for Scotland.
"The referendum in September is creating uncertainty for our customers and our business, which we have a responsibility to address.
"Regardless of the outcome it is critical that we are able to provide continuity of service and protection for their investments and savings.
"To give them full confidence, we have started work to establish additional companies registered in England, in order to provide operational flexibility and to complement our existing business in Scotland."
The company statement highlighted areas of uncertainty for the business, including the jurisdiction and taxation of individual savings and pension plans, financial services regulation and consumer protection.
It also raised concerns surrounding currency and membership of the European Union.
Setting up additional companies registered in England would "remove any uncertainty", the company said
The move would also serve to "provide operational flexibility and to complement our existing business in Scotland", it added.
Alliance Trust, which was incorporated in 1888 from several Dundonian companies, is one of the largest investment trusts in the UK with gross assets of more than £3.2 billion and around 250 employees.
It is the latest in a line of companies to raise concerns about uncertainty created by the referendum.
Generator and heating supplier Aggreko warned yesterday that Scottish independence would lead to ''years of uncertainty and hiatus'' for the business.
Lloyds Banking Group, Barclays, Standard Life, Royal Bank of Scotland, the Macfarlane Group and Breedon Aggregates have all listed independence in their risk management sections of their annual reports.
But other high-profile business figures have voiced support for independence, including Willie Walsh, the boss of British Airways' parent company, who said last month that a Yes vote in September could be a ''positive development''.
A Better Together spokesman said: "What these companies employing thousands of people have in common is that they view walking away from the UK as a huge risk.
"Alex Salmond's failure to set out credible answers on what would replace the pound and our membership of the EU means we are being asked to take a leap into the unknown.
"This is a risk we don't have to take. As part of the UK, we have the best of both worlds, with a strong Scottish Parliament and the back-up of being part of the larger UK economy."
Finance Secretary John Swinney said: "The issues raised by Alliance Trust are entirely addressed by the propositions put forward by the Scottish Government, and show exactly why our proposals for a formal currency area are the right proposals, why they are in the best interests of business on both sides of the border, and why that is what will be implemented by both governments.
"Of course, the real threat to business comes from Westminster's proposed in-out referendum on EU membership, which risks taking Scotland out of Europe, with all the consequences for jobs, investment and prosperity that would entail - and which only a vote for independence will ensure does not happen."
The Finance Secretary also welcomed comments from Mark Wilson, chief executive of Britain's largest insurance firm Aviva, that independence was "not an issue" for the London-based company.
Mr Wilson is reported to have told Sky News: "Obviously we are looking with interest at what's going on but I really think that's an issue for the Scottish people.
"We operate all around the world and we operate in many jurisdictions and in many places, so I really think that's not an issue for us to focus on."
Mr Swinney said: "As Mr Wilson points out, Aviva already operate in many different jurisdictions, which is the reality for most big companies in today's interconnected world.
"Scotland has a strong and diverse economy and the point of independence is to win the powers we need to build on those strengths and create a more prosperous and secure economy - which is good for the financial sector and everyone else."
Blair Jenkins, chief executive of Yes Scotland, said: "Many leading business figures are relaxed about independence because they realise that Scotland, regardless of the outcome of September's vote, will continue to be an excellent place to do business.
"There are also a growing number of leading business figures, including BA boss Willie Walsh, Clyde Blowers founder Jim McColl, and Klin Group chief executive Marie Macklin, who can see clearly the many advantages of a Yes vote."
Aviva later issued a statement clarifying Mr Wilson's comments.
A spokeswoman said: "It was Aviva's year end results announcement yesterday and that was our sole focus.
"When asked about Scotland, Mark Wilson CEO said that we are obviously looking with interest at what's going on, but independence is an issue for the Scottish people.
"He has not said he is 'relaxed' about Scottish independence as some reports suggest.''
Scottish Secretary Alistair Carmichael said: "This has been another week which has injected a real sense of reality into the independence debate.
"People should listen to what Scotland's businesses are saying about independence.
"Standard Life, RBS, Bank of Scotland, Aggreko, the Alliance Trust, BP and Shell are all warning that independence could mean higher costs and moving jobs and headquarters out of Scotland."