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Eastern white pine (Pinus strobus L.) is a widespread conifer in eastern North America. A novel dieback phenomenon, as well as increasing global temperatures contributing to the contraction of suitable habitat, is threatening this species' long-term persistence in the Southern Appalachian Mountains. This southern extent of its current range is where P. strobus is hypothesized to have survived in refugial populations during the last glacial maximum. As a result, extant populations located here may have higher levels of ancestral genetic diversity, and by extension, adaptive potential. We genotyped 432 P. strobus individuals from 23 sites throughout the Southern Appalachians and another 34 individuals from two reference populations in the northern USA, using 10 established microsatellite markers. Levels of genetic diversity in the southern portion of the range were comparable but not higher than reference northern populations. There was an overall heterozygote deficiency and high inbreeding coefficient (F-IS = 0.173); however, these values were comparable to published research of P. strobus throughout the northern range. There was low overall genetic differentiation (F-ST = 0.055) among populations in the Southern Appalachians and population structure was best explained by ecoregions. These results show that P. strobus in the Southern Appalachians is a fairly heterogenous and admixed species with relatively high genetic diversity mostly partitioned within populations. The Southern Appalachians remains an important area for P. strobus conservation, but not necessarily because it is genetically unique.