The purpose of this study was to reveal the differences between ACTN3 genotype (RR, RX, XX) and aerobic performance [Yo-Yo IRT1 (m), VO2 max (ml/kg/min)] in professional and regional amateur league soccer players and to reveal which of these parameters was a distinctive factor in these athletes.71 professional soccer players (age: 23.66 ±4.11 years; body height: 1.79 ± 6.99 m; body weight: 76.02 ± 6.76 kg; body fat: 11.59±3.11 %) and 62 regional amateur soccer players (age: 23.63 ±3.77 years; body height: 1.81 ± 5.77 m; body weight: 76.36 ± 7.53 kg; body fat: 15.60±4.65 %) volunteered for the study. After DNA extraction from buccal epithelial cells via a commercial kit was performed for the genetic background of the athletes, Real-Time PCR was carried out for genotyping. Furthermore, Yo-Yo IRT1 test was performed to determine the aerobic performance of the soccer players. SPSS 23 (SPSS Inc., Chicago, IL, USA) package program was used for the statistical analysis of the data obtained in the tests. Shapiro-Wilk test for normality and Levene's test for homogeneity of variance were performed. Chi-Square, Independent Sample T Test and One Way ANOVA test were used in the analysis of the parameters. Statistical significance was set as p0.05); however, there was a statistical significance in favor of professional soccer players in terms of aerobic parameters (p<0.05). Consequently, it can be said that aerobic performance is the distinguishing factor, not the ACTN3 gene, in soccer players.