ACTN3 gene, which encodes α-actinin-3 and actin-binding protein, has been found to be associated with strong athletic performance, especially among track and field athletes. Therefore, in this study, our aim was to compare the allelic and genotype frequencies of the ACTN3 R577X variant among elite athletes specialized in different branches, and nonathletic controls in Turkey. In thepresent study, 316 subjects, including 168 athletes and 148 sedentary controls were genotyped for the ACTN3 R577X variant. Genotyping was conducted by polymerase chain reaction (PCR) method. Additionally, we evaluated the groups by dividing them as females and males. There were 48 females and 120 males in the athletes group, and 43 females and 105 males in the control group. Genetic associations were evaluated by chi-squire test or Fisher’s exact test. There was a significant difference between the athletes and controls in terms of the ACTN3 R577X variant. ACTN3 RR and XX genotypes increased in the controls compared to the athletes, while RX genotype was higher in the athletes than the controls (P = 0.030). Then we evaluated the groups by separating them as females and males. Genotype distribution of the ACTN3 R577X differed between the male athletes and the male controls (P = 0.046). ACTN3 R577X RX genotype increased in the male athletes compared to the male control (P = 0.046). But ACTN3 R577X genotype and allele distribution was not significant between female athletes and female control group (P>0.05). As far as we know, this study is the largest series examining the ACTN3 R577X variant in Turkish athletes. Our results support that the ACTN3 R577X variant has a heterozygous advantage in athletic performance in the Turkish population. However, epigenetic, gene–gene and gene–environment interactions affects athlete performance should not be forgotten.