The 2021 Lamar Hunt U.S. Open Cup tournament proper was to feature teams from all five tiers of the men's American soccer pyramid.

Qualification for the 2021 tournament was drastically altered due to the ongoing COVID-19 pandemic. The tournament itself was downsized to 16 teams, before ultimately being canceled for the second consecutive season due to the pandemic.

The top eight American-based teams based in the regular season standings after three weeks of the season, taken on a points-per-game (PPG) basis, were set to qualify for the tournament before its cancellation. Qualification began on April 16, 2021, and concluded on May 2, 2021.

The four semifinalists from the USL Championship playoffs qualified. Qualification retroactively began on October 10, 2020, and ended on October 17, 2020.

Phoenix Rising qualify

El Paso Locomotive qualify

Louisville City qualify

Tampa Bay Rowdies qualify