Reading Women were unable to progress into the second round of the FA Cup, losing 3-1 at Aylesford Ladies.
Former semi-finalists in recent years, the move to semi-professional football for the women meant Pedro Bruno's side needed to qualify for the first round.
Brackley Town and Richmond & Kew were defeated in qualifying but the promotion chasers in in Division One of the London & South East League were too much for the Royals.
Kara Richardson opened the scoring for the hosts before captain Jordanne Hoesli-Atkins drew the sides level.
Rebecca Keyte restored Aylesford's lead in first-half stoppage time and Alex Witham put the tie beyond Reading with a converted penalty 15 minutes from time.
This comes on the same weekend Ruben Selles' side beat Fleetwood Town 2-0 in their first round tie to set up a second round tie with Harborough Town.
Reading travel to Tilehurst Panthers in the League Cup on Sunday (2pm kick-off).
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