Mechanical peak power output (PPO) is a determinant of performance in sprint cycling. The purpose of this study was to examine the relationship between PPO and putative physiological determinants of PPO in elite cyclists, and to compare sprint performance between elite sprint and endurance cyclists. Thirty-five elite cyclists (18 endurance; 17 sprint) performed duplicate sprint cycling lab tests to establish PPO and its mechanical components. Quadriceps femoris (QVOL ) and hamstrings muscle volume (HAMVOL ) were assessed with MRI, vastus lateralis pennation angle (PθVL ) and fascicle length (FLVL ) were determined with ultrasound imaging, and neuromuscular activation of three muscles were assessed using EMG at PPO during sprint cycling. For the whole cohort there was a wide variability in PPO (range 775-2025 W) with very large, positive, bivariate relationships between PPO and QVOL (r = 0.87), HAMVOL (r = 0.71) and PθVL (r = 0.81). Step-wise multiple regression analysis revealed that 87% of the variability in PPO between cyclists was explained by two variables QVOL (76%) and PθVL (11%). The sprint cyclists had greater PPO (+61%; P < 0.001 vs endurance), larger QVOL (P < 0.001) and BFVOL (P < 0.001) as well as more pennate vastus lateralis muscles (P < 0.001). These findings emphasise the importance of quadriceps muscle morphology for sprint cycling events.