Starch–gel electrophoresis was used to analyze muscle and liver tissue for variation in 13 enzymes representing 31 presumptive loci in yellow perch (Perca flavescens) from 13 localities scattered throughout the natural geographic range of the species in North America. Ten loci were polymorphic, but only three, alcohol dehydrogenase (ADH-1*), glucose-6-phosphate isomerase (GPI-1*), and phosphoglucomutase (PGM-2*), exhibited polymorphisms at relatively high frequencies across localities. Western populations were fixed for one allele at ADH-1*, eastern populations were fixed for another allele, and populations from intermediate locations in Lake Ontario and Pennsylvania had both alleles. The distributions of alleles at GPI-1* and PGM-2*were similar to that of ADH-1*, exhibiting strong differences between eastern and western populations, although the delineation was not as clear. Western populations were much less variable than eastern populations, and the distribution of alleles indicated that the two groups were derived from Mississippi and Atlantic glacial refugia. Populations near the physiographic discontinuity between the Mississippi and Atlantic drainages in western New York and Pennsylvania exhibited an admixture of typically western and eastern alleles. Such observations are consistent with the mixed faunal history of the region and limited postglacial dispersal of western and eastern populations across the boundary.