The average cost of a one night stay when 24 of America and Europe's best golfers take to the course at Gleneagles will rise to £254 in Perth and £233 in Stirling, the two cities nearest the venue.
The increases, of 155 per cent and 127 per cent respectively, have been criticised by national tourist agency VisitScotland, which has called for a "common sense" approach to hotel charges.
However, there is little chance hotels will lower their costs, with more than 45,000 visitors flooding to see the men's golf competition, which takes place from Friday, September 26 to Sunday, September 28.
Gleneagles is near the small town of Auchterarder, where accommodation options are scarce, so visitors will most likely choose to stay in nearby metropolitan centres, with Perth only 18 miles away and Stirling 19 miles.
The report compiled by Trivago, the online hotel search site, also found hotel prices are set to rise in Dundee, Edinburgh and Glasgow as hoteliers prepare to cater for an overspill of golf tourists.
The average cost of a hotel room in Edinburgh will increase 37 per cent to £172, while Glasgow hotel prices will rise 40 per cent to £138. Booking a room in Dundee, while 40 miles away from Gleneagles, will see fans pay 63 per cent more than usual, with the cost of a single stay now £140 on average.
Denise Bartlett, of Trivago UK, said: "Edinburgh and Glasgow have been in the news recently for their hotel price rises during the Fringe Festival and the Commonwealth Games.
"Despite the criticism, hoteliers in Perth and Stirling appear to be persisting with the price rises, with costs increasing to more than double the amount of the preceding weekend.
"With the Ryder Cup taking place in a relatively isolated location, it is not often Perth and Stirling experience this volume of visitors.
Ms Bartlett added: "Edinburgh, Glasgow and Dundee hoteliers should be commended for raising their prices significantly less than Perth and Stirling - perhaps because they are located slightly further away.
"Our top tip for accommodation is Glasgow, where prices are approximately £100 less than Perth and Stirling and Gleneagles Hotel is just 50 minutes' drive away."
Malcolm Roughead, chief executive of VisitScotland, said it was hoped costly hotel stays would not prevent the Ryder Cup creating a legacy that would see visitors return to Scotland year after year.
He said: "While we cannot directly influence the amount accommodation providers charge, we would urge businesses to adopt a common sense approach over pricing and appreciate that value for money plays a hugely important part in the overall visitor experience whether on business or visiting for pleasure.
"The benefits of the Ryder Cup are set to be felt right across the country's tourism industry from our accommodation sector, through to our visitor attractions and renowned food and drink offering."
Scott Taylor, chief executive of Glasgow City Marketing Bureau, said its hotels had maintained a fair pricing strategy.
He added: "The support from the accommodation industry during the 2014 Games was commendable and has further enhanced the reputation of the city. We welcome the insight from Trivago confirming Glasgow got it right."