Footage of a male pine marten was captured on a wildlife camera in the North York Moors.
The sighting is the first living record in the area for approximately 35 years and the first confirmed record since 1993, when a skull was found.
Pine marten, which look similar to a ferret or stoat but larger, were once widespread in the UK but their population dwindled during the early 20th century and the animal is mainly found in the Scottish Highlands.
Recent DNA tests on droppings have confirmed the animal's presence in Northumberland and there have been a number of unconfirmed sightings and reports of the animals in the forests of Yorkshire.
The Yorkshire Pine Marten Project, run by non-profit social enterprise NatureSpy in partnership with the Forestry Commission, began four years ago, with camera traps set up in various locations around the North York Moors.
The cameras constantly monitor a particular area for months at a time and trigger when an animal passes in front of them, taking a picture or video.
Ed Snell, Yorkshire Pine Marten Project co-ordinator for NatureSpy, said: "To finally prove pine marten presence in Yorkshire is a massive achievement for everyone involved.
"Pine marten are such an important species, being the second rarest carnivorous mammal in the UK, it's so exciting to plan the next stage of the project and aid whatever populations we may have here."
Cath Bashforth, ecologist at the Forestry Commission, added: "It is great to have a confirmed sighting of pine marten on Forestry Commission land.
"Supporting on this project has been exciting and to discover they are living within our forests after so many years is fantastic. We are looking forward to progressing the project further."
The project will now collect DNA samples from individual pine marten, estimate population numbers, look at habitat preferences and produce habitat management proposals.