We examined allozyme variation at 40 presumptive gene loci in 1,904 cotton mice (Peromyscus gossypinus) from 62 locations throughout the range of the species. This species was highly variable: 92.7 percent of loci were polymorphic, observed heterozygosity per population ranged from 0.023 to 0.123 and averaged 0.088, and number of alleles per polymorphic locus averaged 5.6. The least variable populations were from the western part of the range, and the most variable were from the southeastern part. Virtually every population contained unique or rare alleles. Distribution of allozyme variation generally reflected geographic proximity rather than existing subspecies boundaries, with major differences between western, northeastern, southeastern, and insular populations. The most divergent populations were from Georgia and Florida sea islands, but drift alone could not explain this observation, because the largest number of unique alleles per population also occurred in these populations.