Sixteen teams advanced to the second round of the U.S. Open Cup from the A-League/D3 Pro League qualifying. Ten group winners along with six wild card teams advanced. The second round, which will also include all 12 MLS teams and four PDL/USASA qualifiers, is slated for June 27. The A-League/D3 Pro League entrants:
| A-League | D3 Pro League |
| Charleston Battery | Carolina Dynamo |
| Connecticut Wolves | Chico Rooks |
| El Paso Patriots | New Jersey Stallions |
| Hershey Wildcats | Reading Rage |
| Milwaukee Rampage | Utah Blitzz |
| Nashville Metros | |
| Pittsburgh Riverhounds | |
| Richmond Kickers | |
| Rochester Raging Rhinos | |
| San Diego Flash | |
| Seattle Sounders |