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Genetic variation and the allozymic relationships of five families comprising 23 species of hystricognath rodents were examined using electrophoretic data from 19 presumptive gene loci. Direct-count heterozygosity ranged from 0% to 13.6%, and polymorphism varied from 0% to 36.8%. Fixed allele differences were instrumental for the segregation of taxa as summarized by the Distance Wagner procedure and by majority-rule consensus trees. The monophyly of the Octodontidae, and the family rank of the Abrocomidae and Ctenomyidae is supported by the data. Monotypic Myocastor coypus is not resolved as a distinct family but appeared within the Caviidae. Within the Octodontidae, paraphyly was detected in polytypic Octodon, whereas a high bootstrap value supported the node containing the desert specialists O.mimax (2n = 56) and Tympanoctomys barreare (2n = 102). The implications of allozymic data as a robust phylogenetic signal that connected biochemical associations to the prediction of tetraploidy in T. barrerae are discussed.