Rashford was a doubt due to an injury he sustained against Everton in the Premier League.
Now the Three Lions have confirmed he is out of their Nations League matches against Belgium and Iceland this week.
Another who will miss out is Wolves captain Conor Coady.
Coady is self isolating as he came into contact with someone who is positive for COVID-19.
He is taking all the necessary precautions and will not be able to join the national team camp as a result.
Coady told reporters: "I contracted and recovered from the virus myself back in March, and have tested negative twice in camp, but of course it is important that I abide by the rules set by the government and observe a period of self-isolation.
"It's so frustrating to have to miss games because of this, but these are the rules that are set out and the most important thing is to make sure my team-mates are not put at risk."