In this study, we examined gender and age differences in physical performance in football. Thirty-four elite female and 34 elite male players (age 17 +/- 1.6 to 24 +/- 3.4 years) from a professional football club were divided into four groups (n=17 each) according to gender and competitive level (senior males, senior females, junior males, and junior females). Players were tested for specific endurance (Yo-YoIR1), sprint over 15 m (Sprint-15 m), vertical jump without (CMJ) or with (ACMJ) arm swing, agility (Agility-15 m), and ball dribbling over 15 m (Ball-15 m). The Yo-YoIR1 and Agility-15m performances showed both a gender and competitive level difference (P < 0.001). Senior and junior males covered 97 and 153% more distance during the Yo-YoIR1 than senior and junior females, respectively (P < 0.001). Gender but not age differences were found for Sprint-15 m performance (P < 0.001). No difference in vertical jump and Ball-15 m performances were found between senior and junior males (P > 0.05). More marked gender differences were evident in endurance than in anaerobic performance in female players. These results show major fitness differences by gender for a given competitive level in football players. It is suggested that training and talent identification should focus on football-specific endurance and agility as fitness traits in post-adolescent players of both sexes.