Reigning champions Gateshead were knocked-out during the fourth round by Boston United.

[1] Gateshead were the reigning champions for the 2024–25 season, having defeated Solihull Moors in the 2024 FA Trophy final at Wembley Stadium.

[3] Due to the increase in the number of teams in step four, a preliminary round was used for the first time in five years.

[3] The remaining 146 teams from step 4 joined the 14 winners from the preliminary round.

Sittingbourne from the eighth tier is the lowest-ranked team remaining in the competition.