Genetic diversity within and among nine Andropogon gerardii (Big Bluestem) populations from various physiographic regions of North and South Carolina was assessed. Genetic diversity was high at both the species level and at the population level. At the species level, percent polymorphic loci (P) was 96.4% (27 of 28 loci), the number of alleles per polymorphic locus (AP) was 4.07, and genetic diversity (H_e) was 0.425. Mean within population values were P= 82.6%, AP = 2.68, and H_e = 0.351. Within population genetic diversity (He) ranged from 0.190 to 0.466. Allelic richness values per population ranged from 37 to 71. The proportion of genetic diversity among populations (G_st) was 0.166. Mean genetic diversity for the 3 larger populations (He = 0.369) and within the 6 smaller populations (H_e = 0.341) did not differ significantly (P = 0.554). Nei's unbiased genetic identity between pairs of populations ranged from 0.652 to 0.975. Mean genetic identity of individual populations with the 8 other populations ranged from 0.71 to 0.89. A Mantel test showed no significant genetic isolation by geographic distance (r = 0.065; P = 0.614). While banding patterns for most of the loci were consistent with disomic inheritance, two loci (PGI3; UGPP1) displayed patterns consistent with tetrasomic inheritance. Results of this study suggest that Big Bluestem populations in the Carolinas were once more widespread.