Millwall's Under 23s lost 1-0 to Charlton Athletic in the Professional Development League at Greenwich Borough's DGS Marine Stadium.
In a tight derby affair, Jamie Mascoll's first-half injury-time free-kick settled matters.
Next up for Kevin Nugent's side is the return fixture with The Addicks at Calmont Road next Tuesday.
Millwall: Sandford, McNamara, Mbulu, Rooney, Chesmain, White, Wicks, Donovan, Olaofe, Twardek, Brymora. Subs: Hanson, Jackson, Saunders-Henry, Sinclair, Debrah.