The purpose of this study was to present the physiological profile of male soccer players who compete in the professional (Division [D] 1, 2, 3) and semi-professional (D4) Greek soccer leagues, and to compare their physiological profile according to playing division. Using 1,095 players (age: 25.2 ± 4.7 years), twelve anthropometric and physiological characteristics (age, height, body mass, BMI, VO2max, velocity of VO2max velocity at ventilatory threshold, maximum heart rate, maximum lactate, squat jump, 35 m sprint and sprinting fatigue index) were assessed. Factorial analysis of variance revealed a significantly (p < 0.05) enhanced physiological profile amongst the professional, compared to semi-professional players, for 10 of the 12 characteristics assessed between divisions. Regarding aerobic parameters, velocity at maximum oxygen uptake was the variable which discriminated professional, from semi-professional players most. With reference to anaerobic parameters, the 35m sprint was the variable which differentiated players between divisions (i.e. D1/D2 vs. D3/D4). Overall, findings in this study present the physiological profile of soccer players within the specified Greek soccer divisions, with differences identified between professional, and semi-professional divisions. These findings suggest that advanced physiological profiles may contribute to a player’s progression to higher divisions of Greek professional soccer.