The objective of this study is to analyze patterns of genetic variation at genic expressed sequence tag - simple sequence repeats (EST-SSRs) and at chloroplast DNA markers in populations of American chestnut (Castanea dentata Borkh.) to assist in conservation and breeding efforts. Allelic diversity at EST-SSRs decreased significantly from southwest to northeast along the Appalachian range, suggesting repeated founder events during postglacial migration. Comparatively high allelic diversity in Ontario, northwest of the Appalachian range, suggested more recent long-distance dispersal. Clinal variation of allele frequencies along the Appalachian axis was also in accordance with postglacial colonization from one refugium southwest of the Appalachian range. We observed clustering of the northwestern population from Ontario with southwestern populations and sharing of a rare chloroplast haplotype among western populations across the whole latitudinal range. This pattern is consistent with a divergence of postglacial migration routes and higher levels of more recent potentially human-mediated gene exchange between populations west of the Appalachian range. Population pairs east and west of the Appalachian axis showed pronounced allele frequency differences over a small geographic range. These patterns of genetic variation should be considered when sampling reproductive material for conservation and breeding.