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Odontomachus (Latreille) and Anochetus (Mayr) (Hymenoptera: Formicidae: Ponerinae) are closely related pantropical genera of ponerine ants that share morphological and behavioral characteristics. A comparative study was carried out using conventional Giemsa staining, fluorochrome staining, and fluorescent in situ hybridization. Karyotypes revealed a higher stability in chromosome number among Odontomachus species than among Anochetus species. We observed a higher frequency of metacentric chromosomes in the karyotypes of Anochetus compared with the more common telocentrics of Odontomachus species. Differences in the localization of rDNA genes on chromosomes between the two genera also were verified. rDNA genes were found on telocentric and submetacentric chromosomes in Anochetus and on telocentric chromosomes in Odontomachus. Our cytogenetic results lend support to Brown's hypothesis that Odontomachus has evolved from a lineage of Anochetus. The karyotype divergence of both genera can be explained by a model of evolution in which there is a tendency to the increase of chromosome number by centric fission. Supporting evidence for this hypothesis is discussed.