Populations in previously glaciated regions are often genetically depauperate in comparison with populations at lower latitudes, due either to bottlenecks experienced in post-glacial colonization, or to contemporary genetic drift in small, peripheral populations. Populations of the rare self-fertilizing North American orchid Isotria medeoloides are largest in the previously glaciated region near the northern range limit, allowing us to examine the role of historical versus contemporary processes in determining population genetic diversity and structure. If contemporary processes predominate, genetic diversity should increase with increasing census size. In contrast, if sequential bottlenecks associated with colonization are paramount, diversity should decrease with latitude and be relatively insensitive to census size. We genotyped 299 individuals from 20 populations at four variable microsatellite loci to contrast genetic diversity and structure for populations in previously glaciated regions versus previously unglaciated regions. Populations were highly inbred (F = 0.95) and highly differentiated (RST = 0.485). Across all sampled populations, genetic diversity decreased and genetic differentiation increased with declining population size. Small southern populations were especially differentiated and genetically depauperate. In the glaciated part of the range, genetic diversity increased as populations approached the northern range limit, demonstrating the centrality of contemporary processes for this post-glacial colonist.