Genetic variation among nine populations of Ozark chinkapin [Castanea pumila (L.) Mill. var. ozarkensis (Ashe) Tucker], threatened by their susceptibility to chestnut blight (Cryphonectria parasitica (Murrill) Barr), was investigated. Population genetic parameters estimated from isozyme variation suggest the populations have a higher genetic diversity (He = 0.227) than populations of the other Castanea Mill. species on the North American continent, the American chestnut (C. dentata (Marsh.) Borkh.) High levels of heterozygosity were detected within the populations, but nonsignificant differences in genetic diversity were observed among the different populations. Principal component analysis based on isozyme allele frequencies or randomly amplified polymorphic DNA phenotype frequencies showed clustering of the same populations. Populations with high levels of genetic diversity and unusual alleles should be the focal point of conservation biologists for capturing much of the genetic variation of the species.