The demographic and genetic population structure of plains killifish Fundulus zebrinus is presented for fish collected from tributaries of the Kansas and Arkansas river basins in Kansas. Electrophoretic analysis revealed heterozygote deficiencies in fish populations from both basins. These deficiencies are attributed, in part, to a temporal Wahlund effect caused by sampling across length classes of fish of differing genotype frequencies. Individual heterozygosity estimates were not associated with length, sex, or wet weights of fish. However, the proportion of heterozygous genotypes within a river was significantly correlated with the proportion of juveniles collected from the river. This association suggests a relationship between genetic heterozygosity and population fitness in this species. Total mean genic diversity and genetic differentiation among river populations was greater in the Arkansas basin. However, relative genic differentiation among sites within rivers accounted for more of the total genic differentiation in Kansas than in Arkansas basin populations. It is hypothesized that these results may be attributed to decreased gene flow and reduced population densities in the Kansas basin, both effects of relatively greater environmental alteration in this basin than in the Arkansas basin.