English yew (Taxus baccata L.) is a strictly outcrossing and dioecious species whose populations are small and isolated. It is known that sex ratios may vary in natural populations due to local environmental conditions or stochastic events. However, unbalanced sex ratios may have negative impacts on genetic diversity through enhanced genetic drift and inbreeding. The present study represents one of the first attempts to compare the genetic variation at microsatellite loci within and between populations with different gender proportions. Our results indicated that there were no significant correlations between sex ratio and the extent of genetic variation in different populations. All populations exhibited high levels of genetic diversity. Additionally, the genetic structure was characterized separately in male and female individuals. Statistical analyses of the set estimators describing the genetic structure of male and female individuals of T. baccata revealed no significant differences between the two groups. Molecular analysis verified that microsatellite nuclear loci neutrality developed for T. baccata, as there were no significant differences in the genetic variation between males and females and no evidence for any outlier loci using coalescent and hierarchical Bayesian simulations. The results demonstrate that ignoring biased sex ratios in T. baccata populations had no effect on the assessment of genetic differentiation and genetic diversity within and between populations of this species. These results are discussed with regards to the practical application of molecular markers in conservation programs.