Data on multiple paternity within broods has been gathered in several animal species, and comparable data in plants would be of great importance to understand the evolution of reproductive traits in a common framework. In this study, we first isolated and characterized six microsatellite loci from the dioecious plant Silene latifolia (Caryophyllaceae). The polymorphism of the loci was assessed in 60 individual females from four different populations. Two of the investigated loci showed a pattern of inheritance consistent with X-linkage. These microsatellite loci were highly polymorphic and therefore useful tools for parentage analysis. We then used four of the markers to determine paternity within naturally pollinated fruits in four European populations. This study revealed widespread multiple paternity in all populations investigated. The minimum number of fathers per fruit varied from one to nine, with population means ranging from 3.4 to 4.9. The number of fathers per fruit was not significantly correlated with offspring sex ratios. High prevalence of multiple paternity within fruits strongly suggest that pollen competition is likely to occur in this species. This may substantially impact male reproductive success and possibly contribute to increase female and offspring fitness, either through postpollination selection or increased genetic diversity. Wide variation in outcrossing rates may be an overlooked aspect of plant mating systems.