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Genetic variation in stable fly, Stomoxys calcitrans (L.),populations from Nebraska, Canada, and Texas was sampled. Four of 12 allozyme loci were polymorphic, with an average of 1.7 alleles per locus. Observed and expected heterozygosities were 0.086 and 0.070,respectively. Nei's genetic distance between populations averaged 0.001 and ranged from 0.000 to 0.005. Wright's F statistics revealed greater variation within than among populations. Allele frequencies were homogeneous among temporal samples from a single population. Polymerasechain reaction—restriction fragment length polymorphism (PCR—RFLP) analysis of 6.4 kb of the mitochondrial DNA genome with 16 restriction enzymes revealed no variation in stable fly populations from Canada, Nebraska, and Texas. PCR—RFLP analysis of a 2.0-kb fragment of the nuclear ribosomal DNA internally transcribed spacer region also revealed no variation.The lack of genetic differentiation among stable fly populations indicates high levels of gene flow among populations. The low levels of variation observed with biochemical and molecular techniques are consistent with a genetic bottleneck during stable fly colonization of North America.