Tammy Abraham, Ben Chilwell and Jadon Sancho will miss England's game against Wales on Thursday.
The news comes as the Football Association continues to investigate their attendance at a party on Saturday night, which broke the government's coronavirus rules.
Sky Sports News reported that England manager Gareth Southgate is furious that the players have apparently breached the rules.
Sancho and Abraham have apologised and said they will learn from the mistake.
Abraham's friends and family threw a surprise party to celebrate his 23rd birthday at his London flat, with Chilwell and Sancho also there.
Around 20 people were inside the flat, in a clear breach of the national 'rule of six'.
The FA told all three to stay away from St George's Park on Monday, while other England players signed up and had their regular coronavirus test.
The whole squad will be tested again on Tuesday, two days before the Wales game, in line with UEFA's protocols.
But there are no plans for Sancho, Chilwell, and Abraham to be re-admitted into the squad by then, and so - having missed the compulsory tests - there is no way they can be included in Southgate's team to play in the all-British friendly at Wembley.
Southgate warned the squad about breaching the rules after Phil Foden and Mason Greenwood were sent home for leaving the eco-bubble to meet two Icelandic women in Reykjavik four weeks ago.
Southgate has yet to decide if Sancho, Chilwell and Abraham will be allowed back into the England camp in time for the two competitive Nations League games against Belgium and Denmark.
They could join the squad if FA officials arrange for them to be tested on Friday - two days before the Belgium game at Wembley on Sunday.