The Scottish Retail Consortium (SRC) accused the SNP administration of failing to act on business rates, claiming some larger supermarkets are facing bills almost a third higher than similar stores south of the Border.
Director Fiona Moriarty spoke out as figures showed 10.2% of stores in Scotland were vacant in January – up from 9.1% in the same month last year.
The total is lower than the UK national town centre vacancy rate of 10.9%, but that fell from 11.3% in October last year while the Scottish rate increased from 9.9% over the same period.
The closure of a number of high-profile stores – including JJB Sports, Clinton Cards, Comet and Jessops – have led to the loss of thousands of jobs.
Many town centres have become "ghost towns", with boarded-up shop fronts now commonplace in some areas.
However, despite the increase, the Scottish vacancy rate is still significantly lower than that of both Northern Ireland and Wales, where 17.2% and 17% of store premises are empty respectively.
Ms Moriarty said: "It's not so easy to find a chink of light in the Scottish vacancy rate, which has edged up again to the point where more than one in 10 shops are now lying empty in our town centres. If the Scottish Government wants to stem the tide of closures, it should act now to abate the rising costs of doing business on our high streets.
"Unfortunately the Government failed to act on business rates this year, which means that retailers will be hit by an additional £20 million in tax.
"This comes on the back of a cut in empty property rates relief, whilst some larger supermarkets in Scotland are faced with a rates bill which is 28% higher than the same store south of the Border."
The vacancy rate shows no signs of improving in the near future after a warning from experts earlier this year that the number of empty shops could double to reach 40,000.
Retail consultancy firm Local Data Company warned increasing numbers of chain and independent stores are likely to go out of business – leaving behind empty premises as there are no fresh ventures to replace them.
The SRC/Springboard footfall and vacancies monitor also showed shopper numbers in Scotland for January were down 4.6% on the same month last year – the third biggest fall in the UK. It comes after a 6.2% increase in the number of people visiting stores in December.
Ms Moriarty said snow was the "main culprit for deterring Scottish shoppers in January".
She added: "This didn't actually translate into a weak showing for sales. The signs are that people planned well ahead for the bad weather and condensed their shopping into fewer trips."
A Scottish Government spokeswoman said: "The Scottish Government wants to see thriving town centres and is doing all it can to support the retail sector.
"We are introducing measures such as our Fresh Start scheme to bring empty properties back into use, and our town centre review is tackling some of the long-standing issues by working with local councils to put the life back into our high streets.
"Our small business bonus scheme and other reliefs provide zero or reduced business rates for 63% of shops in Scotland."
l The Silverburn shopping centre in Glasgow has secured planning permission for a £20 million extension.
The 200,000 sq ft extension will include nine new restaurants, several redesigned local shops and a 50,000 sq ft cinema, creating up to 350 permanent jobs.