American chestnut (Castanea dentata Borkh.) was a dominant tree species in its native range in eastern North America until the accidentally introduced fungus Cryphonectria parasitica (Murr.) Barr, that causes chestnut blight, led to a collapse of the species. Different approaches (e.g., genetic engineering or conventional breeding) are being used to fight against chestnut blight and to reintroduce the species with resistant planting stock. Because of large climatic differences within the distribution area of American chestnut, successful reintroduction of the species requires knowledge and consideration of local adaptation to the prevailing environmental conditions. Previous studies revealed clear patterns of genetic diversity along the northeast-southwest axis of the Appalachian Mountains, but less is known about the distribution of potentially adaptive genetic variation within the distribution area of this species. In this study, we investigated neutral and potentially adaptive genetic variation in nine American chestnut populations collected from sites with different environmental conditions. In total, 272 individuals were genotyped with 24 microsatellite (i.e., simple sequence repeat (SSR)) markers (seven genomic SSRs and 17 EST-SSRs). An FST-outlier analysis revealed five outlier loci. The same loci, as well as five additional ones, were significantly associated with environmental variables of the population sites in an environmental association analysis. Four of these loci are of particular interest, since they were significant in both methods, and they were associated with environmental variation, but not with geographic variation. Hence, these loci might be involved in (temperature-related) adaptive processes in American chestnut. This work aims to help understanding the genetic basis of adaptation in C. dentata, and therefore the selection of suitable provenances for further breeding efforts.