Abstract. A highly variable mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) locus is used to assess the population structure of mitochondrial genomes in the gynodioecious plant Silene vulgaris at two spatial scales. Thirteen mtDNA haplotypes were identified within 250 individuals from 18 populations in a 20-km diameter region of western Virginia. The population structure of these mtDNA haplotypes was estimated as ST ϭ 0.574 (Ϯ 0.066 SE) and, surprisingly, genetic differentiation among populations was negatively correlated with geographic distance (Mantel r ϭ Ϫ0.246, P Ͻ 0.002). Additionally, mtDNA haplotypes were spatially clumped at the scale of meters within one population. Gender in S. vulgaris is determined by an interaction between autosomal male fertility restorers and cytoplasmic male sterility (CMS) factors, and seed fitness is affected by an interaction between gender and population sex ratio; thus, selection acting on gender could influence the distribution of mtDNA RFLP haplotypes. The sex ratio (females:hermaphrodites) varied among mtDNA haplotypes across the entire metapopulation, possibly because the haplotypes were in linkage disequilibrium with different CMS factors. The gender associated with some of the most common haplotypes varied among populations, suggesting that there is also population structure in male fertility restorer genes. In comparison with reports of mtDNA variation from other published studies, we found that S. vulgaris exhibits a large number of mtDNA haplotypes relative to that observed in other species.Key words. Caryophyllaceae, gene flow, genetic structure, mitochondrial DNA, seed dispersal, sex ratio, spatial structure.Received March 23, 2001. Accepted September 20, 2001 Genetic markers are commonly used to assess the effects of genetic drift and gene flow on structuring genetic diversity within and among populations (Slatkin 1985; Neigel 1997). When it is known that the frequency of genetic markers is influenced by selection, however, that influence on spatial structure must also be considered (Whitlock and McCauley 1999). One such case can be found in gynodioecious plant species in which gender is determined in part by cytoplasmic male sterility (CMS) factors that are located in the mitochondrial genome . CMS factors are maternally inherited and block the production of viable pollen. It has been suggested that the population structure of highly polymorphic mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLP) might reflect both the effects of selection and random processes on the dispersion of CMS genes in natural populations, if these mtDNA variants mark different CMS lineages (Olson and McCauley 2000).Approximately 7% of Angiosperm species exhibit gynodioecious breeding systems in which individuals are either female or hermaphroditic (Richards 1997). It has been known for nearly a century that the genetics underlying sex expression in gynodioecious species can be quite complex, involving an interaction between CMS factors a...