The Reds forward has failed to shrug off an ankle injury in time to face Czech Republic at Wembley on Friday and he will also be absent from the next Euro 2020 qualifier against Montenegro on Monday night.
Despite scoring in the 2-1 Emirates FA Cup quarter-final defeat to Wolves last Saturday, Rashford has been struggling with the problem and is returning to Manchester for more treatment.
“He's been with the medical team all week,” said Southgate at a press conference.
“It's clear to us he's not ready for our matches, so we have sent him back to his club.