Hugh Fearnley-Whittingstall's River Cottage Kitchen in Winchester will remain closed, leaving just one remaining restaurant in Axminster and the group's headquarters in Devon.
A post on the website said: "We all face unprecedented challenges from Covid-19 and with a heavy heart, we have made the very difficult and sad decision that River Cottage Kitchen in Winchester will be closing permanently."
The River Cottage Kitchen in Bristol closed earlier this year, due to "rising costs and challenging market conditions, as well as the size and location of the site". River Cottage Canteen and Deli in Plymouth closed in 2017.
The group's Axminster restaurant reopened on 10 July and in Devon, the 100-acre farm and events space has also reopened.