Development of conservation strategies for Fraser fir (Abies fraseri) in the southern Appalachian Mountains depends in part on recognition of the extent to which Fraser fir is genetically distinct from the closely related balsam (A. balsamea) and intermediate (A. balsamea var. phanerolepis) fir. These sibling species have exhibited intergrading, clinal variation in morphological, chemical, and genetic characteristics in prior research. Chloroplast microsatellite markers were polymerase chain reaction amplified from genomic DNA samples of 78 individuals representing the geographic ranges of Fraser, balsam, and intermediate fir. Gene diversity levels at two loci ranged among taxa from 0.65 to 0.84. Allele frequencies demonstrated significant differentiation among taxa, with R(ST) values of 0.36 and 0.10. Haplotype diversity and D(SH) were highest for balsam fir and lowest for intermediate fir. A haplotype network analysis based on allele size distribution for the two loci revealed two distinct clusters of haplotypes and population-specific haplotypes. Ninety-two percent of the haplotypes in one cluster were from balsam fir and intermediate fir, and 84% of the haplotypes in the other cluster were from Fraser fir and intermediate fir. The genetic differentiation of chloroplast DNA markers provides justification for the recognition of Fraser fir as a distinct Management Unit (MU) for conservation purposes, regardless of its taxonomic classification.