More than 20 bottles, including a Macallan 30 year old, were grabbed in the raid at J L Gill Whisky Shop in Crieff, Perth and Kinross.
It comes just weeks after another break-in at the start of January when two bottles of Macallan 30 year old were taken.
Steuart Cuthbert, whose son Andrew runs the business, said the latest theft has come as another blow.
He said: "Andrew has spent a lot of time building up a business which is now internationally recognised and just when you are beginning to have the world at your feet, people come and knock it away.
"It really is soul-destroying.
"Andrew has become quite a well-known figure in the whisky-collecting community and he has worked extremely hard."
Mr Cuthbert believes the theft was carefully planned as the thieves cut through metal bars to gain access to the shop.
He said: "All of the methods of operation of the break-in indicate that they came prepared.
"They had tools to cut through security bars on the window, it was not an amateur job.
"They cut through the bars about halfway up and wrenched them out of the stonework. Having that sort of equipment in the boot of your car is not really the hallmark of an amateur."
A post on the shop's Facebook page listing the stolen whiskies said: "It has happened again. We have been broken into. Words fail me."
More than 100 people have posted messages of support on the post.
The whiskies stolen included a Macallan 18 year old whisky, Glenfarclas 25 year old quarter cask whisky, a Longrow 18 year old whisky, an Oban 21 year old whisky and a Glengoyne 21 year old whisky while cigarettes were also taken.
It is estimated the whiskies taken are worth at least £10,000 at point of sale in the shop though they could fetch significantly more on the open market.
Although the Macallan 30-year-old can be found selling for £5000 to £10,000 online it was not being sold at such a price in the shop where it was priced at the recommended figure of around £2500.
Officers are appealing for information about the theft which happened between 2.45am and 3.30am on Friday January 25.
A police spokesman said: "Some of these whiskies are exceedingly rare, and consequently very valuable. Lower-value items were left behind, so it appears that those responsible knew what they were looking for.
"It is a good possibility that some of these items have been offered for sale in and around the region, and we would like to hear from you if these items have been offered to you."
Anyone with information is asked to contact police on 101 quoting reference number CR/2146/19 or call Crime Stoppers on 0800 555 111 where anonymity can be maintained.