Rashford and Harry Winks have pulled out of Gareth Southgate’s squad for the Nations League fixtures in Iceland and Denmark.
Grealish has previously represented England Under-21 level and was a member of Southgate’s 2016 Toulon Tournament-winning squad.
It follows call-ups for Conor Coady and Ainsley Maitland-Niles on Saturday.
Following his withdrawal, Man United forward Rashford tweeted: “Gutted. Never want to let this team down but these fixtures just came a little too soon for me.
“I tried my best but I have to focus on starting the season at my strongest for club and country. Good luck boys, I’ll be cheering you on from home.”
England’s players met up at St George’s Park on Monday.
Southgate’s side faces its Euro 2016 conquerors Iceland in Reykjavik on 5 September before playing Denmark in Copenhagen three days later.
The disruption caused to the football calendar by COVID-19, including Euro 2020 being delayed by a year until 2021, means England have not played since November 2019, when a 4-0 win in Kosovo concluded their Euro 2020 qualification campaign.
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