The youth team went down to a 2-0 defeat at home to their counterparts from Scunthorpe United on Tuesday afternoon.
Playing at St George's Park Sheffield Graves, the Spireites caused problems for their opponents in the first half.
Javanne Smith and Matt Fuertado both hit the woodwork and Smith also missed an opening from close range.
Scunthorpe took the lead against the run of play just before the break, scoring with an effort from around 20 yards out.
Mitch Clogg and Alex Netherwood were both sent on to try and spark a revival but Chesterfield failed to level the scores.
With around 15 minutes to go, a defensive error left the keeper exposed, leading to Scunthorpe's second goal.
Reflecting on the performance, coach Neil Cluxton said: "It was disappointing to lose after we started brightly.
"We never really got started in the second half. Tiredness crept in with the players running out of legs.
"We're away at Grimsby on Saturday and we need to regroup a little bit after that."
Spireites: Wharton, Garwood, Gibson, Brook, Emery, Wild, Tansley, Geeves, Smith, Fuertado, Turner
Substitutes: England, Netherwood & Clogg