| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 1 | Cortland St. |
A | 49.20 | |
| 2 | Pitt.-Johnstown |
A | DNS | SFU Red Flash Open |
| 3 | Indiana (Pa.) |
A | 47.84 | Bucknell Team Challenge |
| 4 | Shippensburg |
A | DNS | 2024 PSAC Outdoor Track & Field Championships |
| 5 | California (Pa.) |
A | DNS | 2025 PSAC Outdoor Track & Field Championships |
| 6 | Millersville |
A | 48.00 | |
| 7 | Kutztown |
A | 48.80 | |
| 8 | Lebanon Valley |
A | 49.35 |
| Lane | Team | Squad | Seed | Note |
|---|---|---|---|---|
| 2 | PSU-Harrisburg |
A | 51.72 | 2023 Messiah Multi/Invitational |
| 3 | Messiah |
A | 50.50 | |
| 4 | Gettysburg |
A | 49.43 | Centennial Conference Outdoor Track & Field Championships |
| 5 | Clarion |
A | 50.11 | PSAC Outdoor Track & Field Championships |
| 6 | Dickinson |
A | 51.00 | |
| 7 | Millersville |
B | 52.00 |