Previous analysis of mitochondrial DNA polymorphism in the native range of the European rabbit (Oryctolagus cuniculus) demonstrated the occurrence of two highly divergent (2 Myr) maternal lineages with a well-defined geographical distribution. Analysis of both protein and immunoglobulin polymorphisms are highly concordant with this pattern of differentiation. However, the present analysis of nine polymorphic microsatellite loci (with a total of 169 alleles) in 24 wild populations reveals severe allele-size homoplasy which vastly underestimates divergence between the main groups of populations in Iberia. Nonetheless, when applied to more recent historical phenomena, this same data set not only confirms the occurrence of a strong bottleneck associated with the colonization of Mediterranean France but also suggests a two-step dispersal scenario that began with gene flow from northern Spain through the Pyrenean barrier and subsequent range expansion into northern France. The strength and appropriateness of applying microsatellites to more recent evolutionary questions is highlighted by the fact that both mtDNA and protein markers lacked the allelic diversity necessary to properly evaluate the colonization of France. The well-documented natural history of European rabbit populations provides an unusually comprehensive framework within which one can appraise the advantages and limitations of microsatellite markers in revealing patterns of genetic differentiation that have occurred across varying degrees of evolutionary time. The degree of size homoplasy presented in our data should serve as a warning to those drawing conclusions from microsatellite data sets which lack a set of complementary comparative markers, or involve long periods of evolutionary history, even within a single species.