Current population genetics literature presumes a lack of population structure in microorganisms due to their large population sizes, ubiquitous dispersal, and absence of geographic barriers. Contrary to this, we found evidence of great population subdivision in the social amoeba, Dictyostelium discoideum. Pairwise values of Fst and Rst were significant between samples collected from sites separated by distances from 100m--10,000km (P < 0.05 after sequential Bonferroni). Three- and two-level analyses of molecular variance (AMOVA) confirmed the existence of population structure (Fst 0.30, Rst = 0.48, P < 0.001 for three-level; Fst = 0.297, Rst = 0.474, P < 0.001 for two-level). Further, linearized pairwise Fst values were significantly correlated with natural logarithm of geographic distance (r = 0.471, P < 0.012) indicating isolation by distance. Genotypic diversity of populations remained moderate (Nei (1987) average gene diversity ∼0.50) in spite of the decreased population diversity expected with such Fst and Rst values.