The karyotypes of two subspecies of bat, Macrotus waterhousii Gray (Phyllostomatidae), have been studied and compared. Specimens of M. water¬housii mexicanus from Morelos and Guerrero, Mexico, as well as specimens in one sample of M. waterhousii californicus from Sonora, had a karyotype with a diploid number of 46 including a telocentric Y-chromosome in the male complement. An¬other sample of M. waterhousii californicus from Sonora, as well as samples from Arizona and California, had a diploid number of 40. Of these samples, the Arizona specimens also revealed a telocentric Y-chromosome and in addition a secondary constriction on the supposed X-chromosome, whereas the California sample was characterized by a pronounced achromatic region near the centromere on one of the large pairs of telocentric chromosomes and the existance of a meta or submetacentric Y-chromosome in the male. This appears to be the first major karyo type variation of bats reported at the subspecific level.