Miguel Llera's youth team suffered a 1-0 defeat away to Rotherham United yesterday, in the EFL Youth Alliance North East.
A single goal was enough to settle the contest at the Roundwood Sports Complex, and brought to an end the Spireites' two-match winning streak.
Miguel Llera's side are next in action tomorrow (Saturday), when they take on Mansfield Town at St George's Park, Sheffield Graves, 11am.
Chesterfield XI vs Rotherham: Marris, Howard, Willock, Sharman, Render, Hand, Feurtado, Ofoegbu, Holmes, Amantchi, Rawson.
Substitutes: Emery, Turner, Sennett-Neilson, Smith, Gibson.