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<it>Biochemical polymorphism and systematics in the genus</it> Peromyscus. <it>V. Insular and mainland species of the subgenus</it> Haplomylomys. <it>Syst. Zool. 23:226–238</it>.—We have examined allozymic variation at 25 loci in nine species of <it>Peromyscus</it> inhabiting the southwestern United States, Sonora, Baja California, and islands in the Gulf of California. Four previously studied species of <it>Peromyscus</it> are also included in a dendrogram formed by cluster analysis of genie similarity coefficients. Mainland populations currently assigned to <it>P. eremicus</it> represent two distinctive genetic types, an eastern form in Nevada, New Mexico, Arizona, Texas, and Sonora, and a western form in southern California and Baja that may have been separated originally by the Gulf of California embayment in the Pleistocene. <it>Peromyscus merriami</it> is genically distinct from sympatric <it>P. eremicus</it> populations of the eastern type, although it falls within the range of genetic variation found between eastern and western <it>P. eremicus</it> forms. The insular endemics <it>P. guardia, P. interparietalis</it>, and <it>P. dickeyi</it>, and two insular subspecies of <it>P. eremicus</it>, are similar to the western <it>P. eremicus</it> type on Baja and probably share a recent common ancestor. Populations on shallow-water islands near Baja are more similar to mainland populations than are those on deep-water islands. Other insular species, <it>P. caniceps</it> and <it>P. stephani</it>, are genetically more distinct from <it>P. eremicus</it> and may have closer relationships with other mainland species. <it>P. sejugis</it> is closely related to <it>P. polionotus</it> (<it>maniculatus</it> species group) and probably evolved from <it>maniculatus</it>. There is considerable variation in level of genie heterozygosity among mainland populations, although the mean of six percent is consistent with “normal” heterozygosity estimates for other vertebrates. All insular populations have low variability, averaging less than one percent of loci in heterozygous state, presumably as a consequence of random drift in small populations.