Little is known about the population structure of social microorganisms, yet such studies are particularly interesting for the ways that genetic variation impacts their social evolution. Dictyostelium, a eukaryotic microbe widely used as a developmental model, has a social fruiting stage in which some formerly independent individuals die to help others. To assess genetic variation within the social amoeba Dictyostelium purpureum, we sequenced ∼4000 base pairs of ribosomal DNA (rDNA) from 37 isolates collected in Texas, Virginia, and Japan. Our analysis showed extensive genetic variation between populations and clear evidence of phylogenetic structure. We identified three major phylogenetic groups that were more different than other accepted species pairs. Tests using pairs of clones showed that both sexual macrocyst and asexual fruiting body formation were influenced by genetic divergence. Macrocysts were less likely to form between pairs of clones from different groups than from the same group. There was also a correlation between the genetic divergence of a pair of clones and their degree of mixing within fruiting bodies. These observations suggest that cryptic species might occur within D. purpureum and, more importantly, reveal how genetic variation impacts social interactions.