Boots has said 4,000 jobs will go, while John Lewis is shutting down eight stores, putting 1,300 jobs at risk.
The moves come amid warnings that new economic support from Chancellor Rishi Sunak will not be enough to stop millions of workers losing their jobs.
Mr Sunak admitted that he would not be able to protect "every single job" as the UK enters a "severe recession".
Boots is consulting on plans to cut head office and store teams and shut 48 of its more than 600 Boots Opticians practices.
It has not yet said which outlets will close, but about 7% of its workforce will lose their jobs.
John Lewis said department stores in Birmingham and Watford will not reopen as the coronavirus lockdown eases. It also plans to shut down its At Home stores in Croydon, Newbury, Swindon and Tamworth and travel sites at Heathrow airport and London St Pancras.
Mr Sunak unveiled a series of measures on Wednesday aimed at saving jobs, including a one-off £1,000 payment to employers for every furloughed employee retained to the end of January 2021.
He also announced measures to benefit the hospitality sector, including giving diners 50% off eating out from Monday to Wednesday in August.
Culture Secretary Oliver Dowden said the moves to support restaurants, pubs and cafes could also help retail.
"We very much hope that when people go to their local pub or their restaurant to eat out, those are often in the centre of towns, hopefully that will encourage the footfall to those areas so we get more people going to our shops as well," Mr Dowden said, speaking after announcing the reopening of gyms, indoor pools and outdoor theatres.
ohn Lewis and Boots are the latest in a long line of companies to have made cuts during the pandemic. Other lay-offs announced include:
Up to 5,000 job cuts at Upper Crust owner SSP Group
Up to 12,000 jobs at British Airways
Up to 700 jobs at Harrods
About 600 workers at shirtmaker TM Lewin
1,900 jobs at Café Rouge-owner Casual Dining Group
1,000 jobs at Pret A Manger
1,700 UK jobs at plane-maker Airbus
1,300 crew and 727 pilots at EasyJet
550 jobs are going at Daily Mirror publisher Reach
'Jobs loss tsunami'
Unions and analysts have warned that the virus could mean millions of people end up out of work, warning that government incentives to save jobs were not large enough to persuade bosses to keep workers.
Len McCluskey, general secretary of the Unite union, said: "With no modification to the jobs retention scheme, that dreaded October cliff-edge for businesses and workers has now been set in stone.
"Our fear is the summer jobs loss tsunami we have been pleading with the government to avoid will now surely only gather pace."
Vivienne King, chief executive at Revo, which represents the retail property sector, warned that three million retail jobs remained in jeopardy unless the government undertook "a fundamental review of business rates and direct financial support to underwrite rents".
Chancellor Rishi Sunak himself told BBC Breakfast: "Is unemployment going to rise, are people going to lose their jobs? Yes, and the scale of this is significant.
"We are entering one of the most severe recessions this country has ever seen. That is of course going to have a significant impact on unemployment and on job losses."
Lucy Powell, shadow minister for business and consumers, said the job cuts were "deeply worrying news for staff at John Lewis and Boots" and described Mr Sunak's statement as "a missed opportunity to protect jobs with properly targeted support for the businesses and people that need it".
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