The retailer said the concept had already been successfully tried out at a store in Leeds, and it now plans to stock the used garments more widely.
For the venture, the George at Asda brand has joined forces with specialist wholesaler Preloved Vintage Kilo.
The move comes after Asda launched a scheme encouraging customers to take unwanted clothes back to stores.
Asda said the latest scheme would "give a new lease of life" to pre-worn garments.
It would enable customers to "buy vintage, retro and second-hand branded pieces, preventing thousands of tonnes of garments going to landfill each year".
Retailing under the PVW (Preloved Vintage Wholesale) brand name, the second-hand clothes will be available in stores in London, Bristol, Birmingham, Edinburgh and Brighton, among others.
Asda is the UK's third biggest supermarket chain with 632 stores. In February it was sold for £6.8bn to the Issa brothers, two entrepreneurs from Blackburn, and the investment firm TDR Capital.
Preloved Vintage managing director Steve Lynam said the company had prevented more than 800 tonnes of clothing ending up in landfill and that linking with Asda would increase that "dramatically".
"In a world where we are becoming more environmentally conscious this partnership will help bring sustainable fashion to the mainstream which is something as a business we strive for in everything we do.
"The more people that buy into the circular economy and shop vintage & retro the bigger impact we will have on climate change."
The issue of sustainable fashion has been embraced by a number of retailers in recent years.
Several companies take back second-hand clothes, including Asda, Primark and M&S, which have recycling scheme which allow customers to return used items in stores.
In 2019, upmarket store chain Selfridges teamed up with the resale site Vestiaire Collective to sell on second-hand garments.
Online sites which provide a platform for users to sell on unwanted clothes include the Depop social shopping app which was established in 2011 and more recently Vinted and Asos Vintage.
The trend is not confined to clothing. Earlier this year home retailing giant Ikea announced it would press ahead with its plans to buy back and resell used furniture.