Los Angeles FC is the defending champion, having won the 2024 edition.
[3] The 16 direct participants were determined by their non-qualification for other competitions or their placement in the 2024 Supporters' Shield standings.
[6][7] Half of the MLS entrants in the Fourth Round will host their matches, while the rest will play away.
U.S. Soccer also adopted new rules for the competition that restricts the use of alternative venues and has random draws for hosting beginning in the Round of 16.
[8] Metro areas with multiple teams: A total of 36 states and D.C. were represented by clubs in the U.S. Open Cup this year.