| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | Central Florida Christian |
A | Cherubin | Mack | Munroe | Reed | NT | |
| 3 | Lake Brantley |
A | McDonald | Collier | Taylor | Perry | 44.16 | FHSAA 4A District 3 |
| 4 | Apopka |
A | Kemp | Navilsaint | Jarman | Gibbs | 44.43 | Winter Park - Wildcat Open 2026 |
| 5 | University (OC) |
A | Burns | Davis | Andrews | Santos | 45.84 | Hagerty Track and Field Invitational 2026 |
| Lane | Team | Squad | Athlete 1 | Athlete 2 | Athlete 3 | Athlete 4 | Seed | Note |
|---|---|---|---|---|---|---|---|---|
| 2 | DeLand |
A | Carey | Dumas | Jackson | Nicholson | 43.45 | 22nd Annual East Coast Classic |
| 3 | Lake Howell |
A | Spells | Patterson | Funnye' | Wilkerson | 42.23 | Ocoee Relays |
| 4 | Hagerty |
A | Painter | Groenink | Richburg | Richburg | 41.78 | FHSAA State Track & Field Championships (4A) |
| 5 | Lake Mary |
A | John | Williams | Simmons | Dixon | 42.08 | FHSAA 4A District 2 |
| 6 | Mainland |
A | Estes | WILLIAMS | Holmes | Berry | 42.40 | FHSAA 3A District 6 |
| 7 | Olympia |
A | Smith | Robinson | Rathmann | Toledo | 43.68 | FHSAA 4A District 5 |