Red Squirrels in South of Scotland is increasing its trappers to 10 targeting hotspots along the border in a bid to cull disease-carrying grey squirrels crossing from England.
The group is working with conservationists in Cumbria and Northumberland, and is taking heart from effective culling programmes there.
Buccleuch Estates communications director Richard Williamson said the need for action was critical.
"It was either do something radical now or we will be writing off the red squirrel in Scotland."
The grey squirrel, originally from America, is immune to the squirrelpox virus but it is fatal to the native red squirrels.
Mr Williamson said: "It's lethal to reds and it's a really unpleasant death for them."
Traditionally the greys in Scotland have been free from the disease but in May this year a disease-carrier was trapped at Floors Castle, near Kelso. It was followed by a diseased squirrel being discovered at Drumlanrig Castle in Dumfriesshire.
The project, from Arran in the west through south Lanarkshire to the Borders, pulls together the work of three groups which have been fighting the grey invasion.
The steering group for the new initiative met for the first time last week.
"We recognised the only way we can save – literally save – the red squirrel population in the south of Scotland is by having a concentrated cull of grey squirrels," said Mr Williamson.
The Scottish Wildlife Trust and Scottish Natural Heritage-led project brings together local estates, including Buccleuch, Roxburghe, Lothian and Torwoodlee as well as the Forestry Commission.
"Red squirrel conservation is important but our efforts are now being targeted at halting the spread of the disease, " said Mr Williamson.
Specific areas for increased trapping are along the A7 corridor and rivers from Langholm to Hawick. Mr Williamson said the possible next hotspot would be the Tweed Valley around Innerleithen and Peebles.
Four Forestry Commission rangers trap already, there will be two from the Scottish Wildlife Trust, some are from the Southern Uplands Partnership and paid by Scottish Natural Heritage and others are gamekeepers in the private sector.
Those not already involved are being recruited now and will start work at the beginning of next month.
"We're aiming for a cordon sanitaire. Trapping has been carried out but this is the first time it's been co-ordinated in this way.
"Our aim is to halt the spread of the disease. We are trying to stop the infected greys coming up and getting into the Scottish central belt population because then the pox would run riot up north into the reds.
"As Scotland holds 75 per cent of the UK population, it is vital we keep the population here viable," said Mr Williamson.
"We won't eradicate the grey squirrel in the south of Scotland but our aim is to reduce the population as far as possible."
The organisation will launch a red squirrel hotline soon and will urge members of the public to report grey sightings so trappers can be deployed.
He said: "We have got a huge bank of support in the south of Scotland. People here are very pro red squirrels and the public very much has a part to play."
Red squirrel conservation officer, Richard Wales, the author of the new project, said: "It's fantastic we have got what we have been trying for for so long.
"There's a huge amount of private sector support and by utilising the public support, it's very much a bottom up approach as opposed to the top down.
"We are currently creating a buffer zone from coast to coast. We need to make sure we keep the pox out.
"If the pox gets north of the border we are going to see the extinction of the red squirrel in the UK."