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All short-term let (STL) accommodation must have a licence and those who fail to apply can be fined up to £2,500.
The deadline has already been pushed back by six months to 1 October, but some in the sector have asked for more time.
STL operators have warned the new rules could negatively affect the economy, tourism and jobs.
According to the BBC, the Scottish Bed and Breakfast Association (SBBA) has urged the first minister to delay the scheme, arguing it will hit tourism and jobs.
The first minister said:
"There will not be another extension to the deadline.
"It is the right thing to do to bring this licensing scheme in. There has, of course, been an extension already.
"We continue to work with the sector, in fact cabinet secretary Neil Gray met with the sector, I think it was just last week.
"We'll continue to engage with the sector where we can but there'll be no more extensions."
Fiona Campbell, chief executive of the Association of Scotland's Self-Caterers, said the legislation "has the potential to make Scotland a laughing stock".
"A country inflicting harm to its economy, businesses and its people, all the while confused about why it's doing so."
"It's disappointing, and deeply saddening, that the first minister is digging his heels in, as small tourism businesses across the country prepare to close their doors and worry about how they will put food on the table."
"After the shambles of DRS and plans to ban alcohol marketing, the Scottish government made a virtue of its desire to engage with industry and listen to their concerns. We foolishly believed them.
"This policy is threatening the livelihood of countless small businesses - bed and breakfasts, self-catered cottages and the restaurants, cafes, taxis and other businesses supported by the tourism they bring.
"Meanwhile, corporately owned aparthotels are exempt from the SNP and Greens plans."