The sex ratio is one of the most intriguing problems for evolutionary biologists. Spatio-temporal variation of male frequency in sexually dimorphic plant species, spatial segregation, and differential investment of the two sex functions have attracted much research interest. In this study, we examined altitudinal variation of the sex ratio and segregation by gender in Mercurialis perennis (dog’s mercury), a dioecious anemophilous species with wide distribution. The eight studied populations from Serbia represented an altitudinal range of 196 to 1480 m. Sex ratio was significantly biased in seven of the eight populations, with males outnumbering females 3.91:1 in the lowest-altitude population. Our results support the notion of spatial segregation of sexes along on altitudinal gradient: the frequency of males decreased with altitude, from 79.6% to 41.0%. The sex ratio was not significantly correlated with population density. We also examined intersexual differences of plant height in two populations from the extremes of the studied altitudinal range. Males were the larger sex in both populations, though the difference was not significant in the high-altitude population. .