The purpose of this study was to examine muscle characteristics of the vastus lateralis (VL) and body composition of National Collegiate Athletic Association (NCAA) Division I football players. Sixty-nine Division I football players (mean ± SD; age: 20.0 ± 1.1 years; height: 186.2 ± 7.0 cm; body mass: 106.3 ± 21.1 kg; %fat: 17.8 ± 4.6%) were stratified by player position, race, year, and starter status. A panoramic scan of the VL was performed using a GE Logiq-e B-mode ultrasound. Muscle cross-sectional area (mCSA) and echo intensity (EI) were determined using Image-J software from the VL scan. Body composition measures were determined using dual-energy x-ray absorptiometry (DXA). For mCSA, defensive linemen (DL: 46.7 ± 4.2 cm) had significantly greater CSA (p ≤ 0.05) than wide receivers (WR), linebackers (LB), defensive backs (DB), punters/kickers (PK), and running backs (RB). There were no significant differences for EI (p > 0.05) between positions. Offensive linemen and DL had significantly greater %fat than WR, LB, DB, PK, and RB (p ≤ 0.05); greater lean mass than all other positions (p ≤ 0.05); and more fat mass than quarterbacks, WR, LB, DB, PK, and RB (p ≤ 0.05). There were no muscle or body composition differences for race, year, or starter status. Because no differences between positions were observed for EI measures, it may indicate that competitive athletes have increased muscle quality regardless of body composition differences. Ultrasound and DXA measures may be useful to identify muscle characteristics and imbalances if a player gains or loses weight, suffers an injury, or declines in performance.