Geographic protein variation in 26 genetic loci from six samples of Pseudacris brimleyi was analyzed by standard and sequential starch-gel electrophoresis. A single population of P. feriarum was included as an outgroup. In these seven populations, standard methods detected 59 alleles while sequential analysis revealed 31 additional alleles at 14 loci, a 53% increase over the number of alleles using standard methodology. Mean heterozygosity in P. brimleyi increased from 8.5% to 12.0% and the percentage of polymorphic loci increased from 26% to 34%. The four northern populations of P. brimleyi from North Carolina and Virginia are all quite similar to each other genetically, while the two from South Carolina are more differentiated. These results are consistent with the hypothesis that P. brimleyi may have recently expanded its range northward.