Pine marten populations have been detected south and west of Glasgow as well as in the Upper Tweed Valley and Annandale and Eskdalemuir in eastern Dumfries and Galloway.
A report by Scottish Natural Heritage (SNH) and the Vincent Wildlife Trust (VWT) confirmed their presence in three new separate areas of southern Scotland.
Pine martens were once found throughout the UK but suffered one of the most dramatic declines of any UK mammal. Woodland clearance, trapping for fur and predator control by gamekeepers led to a widespread decline in the 19th century.
Populations have recently recovered in Scotland and are now established in most areas north of the Central Belt, but are still rare in the UK and absent from most of England and Wales.
Numbers in Scotland are estimated to be between 2600 and around 3500.
A small number were re-introduced to the Galloway Forest in the early 1980s, but the new arrivals are not thought to have spread from this group, which has remained in isolation. The new groups have most likely originated from a mix of natural spread and deliberate releases.
The information comes from a survey in which pine marten droppings were collected from woodlands and records were collected from foresters, naturalists and local record centres.
SNH said there was not yet enough information for population estimates.
Lizzie Croose, the VWT's survey coordinator, said: "Pine martens have been absent from most of southern Scotland for almost 200 years, so their return is significant."
Rob Raynor, SNH's mammal advisory officer, said: "At present, re-colonisation of the new areas is still at an early stage, but if breeding populations do establish successfully, pine martens will probably expand throughout southern Scotland and south into northern England."