Genomic DNA was compared between three typical species of rodent mycoplasmas, Mycoplasma pulmonis, M. arthritidis and M. neurolyticum, and between strains of these species. Each of the three species showed a distinct restriction endonuclease cleavage pattern of genomic DNA. The genetic heterogeneity of these species was revealed by total DNA hybridization as well. In addition, the restriction endonuclease cleavage pattern of genomic DNA was almost identical in three strains of M. pulmonis and in two strains of M. neurolyticum. The genetic hemogeneity among strains of the same species was revealed by total DNA hybridization as well. These data suggest that the genomic DNA sequence of each rodent mycoplasma species has a high degree of species specificity.