Wayne Rooney and Derby's entire first-team squad will miss Saturday's FA Cup tie against Chorley following the coronavirus outbreak which has closed their training ground, it is understood.
The Rams will instead field a team made up of under-23 and under-18 players at Victory Park on Saturday afternoon.
A number of first-team players and staff returned positive tests for Covid-19 on Monday, with the club's training facilities subsequently closed.
Derby issued a statement on Monday, confirming the closure of their training ground, and saying the club was "in dialogue with the EFL and Football Association in relation to upcoming fixtures".
FA Cup rules state that clubs are expected to fulfil their fixtures if they have 14 eligible players - including from the under-23 and under-18 squads - available, though there is scope for appeal.
The fixture at Chorley's Victory Park has been selected for live TV coverage and is scheduled to kick-off at 12.15pm on Saturday.
The news comes on the same day that it was announced the EFL will test players from all 72 of its clubs for twice a week from Monday after the Professional Footballers' Association agreed to fund the programme.
The competition has been beset by a raft of postponements related to Covid-19 in recent months which have led to calls for it to introduce a regular testing regime or risk the league being suspended.
The league had committed to testing at 'strategic' points of the season – which include this week – and clubs could test more regularly at their own discretion.
But this move brings uniformity and a greater degree of certainty at a time when a number of clubs have called for a "circuit breaker" with a new, highly transmissible strain of coronavirus in circulation.
Lateral flow devices will be used to test all players and staff, and any individual who tests positive will be required to undertake a polymerase chain reaction (PCR) antigen test.
Two sets of results will be announced each week confirming the number of individuals tested and the number of positives.