The prevalence of varus knee malalignment among junior and adult football players (FP) has proven to be higher compared to other sports. No causal relationship has yet been found, as genu varum can be assumed to be an independent risk factor for the development of knee osteoarthritis. The purpose of this study is to compare knee alignment measurements and sport-specific data of adolescent football players and referees (REF). Knee alignment was detected by measuring the intercondylar/intermalleolar distance (ICD/IMD) as well as the Hip-Knee-Ankle angle (HKA) using a standardized digital frontal-plane photograph. Anthropometric and sports-related data (training/match exposure, seasons actively played, etc.) were collected by means of questionnaires (Clinical trial registration number: DRKS00020446). A total of 28 male FP and 29 male adolescent REF were included in the survey. The mean age was 17.4 & PLUSMN; 0.7 years. The two groups did not differ significantly in age, height, weight, BMI, and overall football/refereeing exposure per week (FP vs. REF: 274 vs. 285 min/week, p = 0.61). The HKA of the FP was significantly lower (toward varus) than that of the REF (177.6 & DEG; & PLUSMN; 2.4 & DEG; vs. 179.0 & DEG; & PLUSMN; 2.4 & DEG;; p < 0.001). However, ICD did not significantly differ (FP: 17 & PLUSMN; 25 mm, REF: 13 & PLUSMN; 27 mm; p = 0.55). The football environment with frequent football exposure seems to have an influence on leg axis deviation in FP compared to REF. For prevention of knee osteoarthritis in FP, an advanced understanding of leg axis development in adolescent players is essential and, therefore, needs further research.