Grealish comes in with Gareth Southgate unable to select Mason Mount due to the Chelsea midfielder isolating until next Monday.
Mount and Ben Chilwell were confirmed as a close contacts of Scotland midfielder Billy Gilmour, who tested positive for Covid-19 earlier this week.
Maguire makes his first start since 9 May, having recovered from an ankle ligament injury which led him to miss the end of Manchester United’s season.
Saka has got the nod to start ahead of Jadon Sancho and Marcus Rashford and replaces Phil Foden, who would have been suspended for England’s last-16 tie had he received a yellow card.
The Three Lions must win tonight in order to top Group D ahead of Czech Republic, who hold a superior goal difference as the tie breaker.
England XI: Pickford; Walker, Stones, Maguire, Shaw; Phillips, Rice; Saka, Grealish, Sterling; Kane.
Substitutes: Ramsdale, Johnstone, Henderson, Rashford, Trippier, Mings, Coady, Sancho, Calvert-Lewin, White, James, Bellingham.
Czech Republic XI: Vaclik; Coufal, Kalas, Celustka, Boril; Soucek, Holes; Jankto, Masopust, Darida; Schick.
Substitutes: Mandous, Koubek, Kaderabek, Brabec, Barak, Krmencik, Sevcik, Hlozek, Vydra, Kral, Mateju, Pekhart.