Introduction: Genetic factors appear to explain why some athletes perform better in competition and training than their peers. Objective: To determine the occurrence of R577X polymorphism of the ACTN3 gene in mountain runners. Methods: The sample consisted of 19 female mountain runners with a mean age of 41.2 ± 6.1 years. Genotyping of R577X polymorphism of the ACTN3 gene was performed by the polymerase chain reaction (PCR) method with DNA extracted from saliva. The genotypic and allelic frequencies of the athletes were evaluated and compared with data from the literature. Hardy-Weinberg equilibrium and Chi-square with Yates correction were used, with a significance level of p<0.05. Results: The genotypic distributions did not show any significant differences between the athletes and the control group, with RR = 15.8%, RX = 57.9%, XX = 26.3%. In regard to allelic distribution, the nonfunctional allele was higher in the study group than in the control group, with R = 44.7%, X = 55.3% for p = 0.0350. Conclusion: The data revealed a possible relationship between the ACTN3 X allele and athletic performance in Brazilian female mountain runners. Level of evidence II; Development of diagnostic criteria in consecutive patients (with “gold” reference standard applied).