Background: The X-allele of the α-actinin 3 (ACTN3) R577X gene polymorphism has been positively associated with elite endurance athlete status [1, 2] although others have found no association [3, 4]. The aim of the study was to: (i) compare the ACTN3 R577X genotype and allele frequency distributions in ‘elite’ and ‘sub- elite’ marathon runners with those of a non-athlete population; (ii) determine whether marathon personal best time (PB) was associated with ACTN3 R577X genotype. Methods: One hundred and twenty-four elite (men n=31; women n=31) and sub-elite (men n=43; women n=19) athletes, who participated in the 2013 London Marathon, and 566 non-athlete controls provided whole blood, buccal or saliva samples, from which DNA was isolated and the ACTN3 R577X polymorphism genotyped. PBs were verified via www.powerof10.org and used to determine elite (men <2.5 h; women <3 h) or sub-elite (men 2.5-3 h; women 3-3.5 h) status. Chi-square analysis was used to compare genotype and allele frequency distributions between athletes and controls, while any genotype-dependent differences in marathon PB were investigated using a one-way ANOVA for men and for women.Results: There were no significant differences in genotype or allele frequency distributions between the elite (RR 35.5%, RX 53.2%, XX 12.9%; R 61.3%, X 38.7%) and sub-elite (RR 29.0%, RX 53.2%, XX 17.7%; R 55.7%, X 44.3%) or controls (RR 35.2%, RX 47.5%, XX 17.3%; R 59.0%, X 41.0%); P ≥ 0.393. PBs were not genotype-dependent for either men or women (P ≥ 0.314).Conclusions: The findings strongly suggest that the ACTN3 R577X polymorphism does not play a role in influencing elite/sub-elite marathon runner status, or in determining marathon PB performance.