Peter Hughes, chief executive of Scottish Engineering, said the "vast majority" of his members wanted the vote to be held before autumn 2014, the SNP's preferred timescale. It followed a survey of members of the group, which indicated only 22% were happy with the 2014 date, while almost three-quarters (74%) wanted the referendum to be held "much earlier".
Mr Hughes said uncertainty over the issue could undermine company development both in Scotland and abroad and he called on politicians to answer key questions on independence as he accused them of indulging in "political rhetoric".
His intervention, in the latest issue of the group's quarterly review which is published today, follows similar calls from industry leaders, including SSE and the Weir Group, in recent days. Yesterday the chief executive of the Miller Group, the Edinburgh construction and property firm, also called for the vote to be held "sooner rather than later".
Mr Hughes's comments were welcomed by Scottish Secretary Michael Moore, who said business was making its voice heard "loud and clear". He said an "undeniable head of steam" was building up, adding: "Let's get on with asking the Scottish people a single question next year."
But the Scottish Government hit back last night, claiming other industry leaders had said that holding the vote in 2014 would make no difference to business.
Finance Secretary John Swinney also insisted his administration was "absolutely committed" to having a full debate on Scotland's future.
The row comes as Labour leader Ed Miliband is today expected to tell the Scottish Labour conference in Dundee that Scotland and England would be weaker apart, saying: "If we are going to build an economy that works for working people, we are going to have to build it together."
Earlier this week it was claimed the referendum would be held on October 18, 2014, although this was later played down as only a possibility. It is understood the Scottish Government's preferred timescale is a three-week window around that date.
The Scottish Engineering survey analysed about 100 responses from the group's 400 members and Mr Hughes said results indicated most were keen to see a "speedy resolution" to the referendum issue.
He added: "As we all know, uncertainty can seriously undermine the confidence of companies growing and developing their business both at home and abroad.
"As yet we have not seriously entered the well-informed debate phase on the question of independence. It is important our politicians start to answer the key questions which we have been asking, rather than indulge in political rhetoric."
Mr Swinney said the executive director of the Institute of Directors (IoD) Scotland, David Watt, had said holding the referendum in 2014 would make no difference to business and none of the IoD's 1600 Scottish members had raised any concerns about the timetable.
Mr Swinney said: "We are absolutely committed to having a fully informed debate on Scotland's future, which is why in the coming months we will continue to set out what independence means for people and business in Scotland."
He added that the Government would publish the full prospectus for independence in autumn next year.