We developed 12 highly polymorphic microsatellite markers for the eastern box turtle (Terrapene carolina carolina). The number of alleles per locus ranged from 5 to 27 (mean: 15.2 per locus) and the observed heterozygosities ranged from 0.371 to 0.943 among 35 individuals. Two loci exhibited significant deviations from Hardy–Weinberg equilibrium. Five loci exhibited putative null alleles but this is probably due to the high number of alleles relative to the sample size. There was no evidence of linkage disequilibrium between pairs of loci. This library should facilitate research on the population structure and ecology of this threatened species.