The DNA karyotypes of three species and several subspecies of New World Leishmania were found to be distinct. The karyotypes were more similar among closely related isolates than among more distantly related groups. Two classes of chromosomal DNA differences were detected among stocks; ± 50 kb size differences among DNAs, some of which were shown to contain homologous sequences, and DNAs having no obvious corresponding chromosomal DNA in other isolates. A total of 14–24 chromosomal DNA bands were resolved, depending on the isolate, but densitometric analyses suggest that these isolates contain 26–33 distinct DNA molecules. These molecules total about 2.5 × 10⁷ bp, a substantial fraction of the genomic DNA. The chromosomal DNA locations of gene sequences homologous to α- and β-tubulin, ribosomal RNA, thymidylate synthetase-dihydrofolate reductase, and the H-region sequence were determined. The homologous sequences were located on chromosomal DNAs of similar, but not identical sizes among different stocks. We also found species- and some subspecies-specific β-tubulin chromosomal loci. We conclude that the DNA karyotype is useful for stock identification, taxonomy, and gene localization in Leishmania. Its potential for identifying the species and subspecies in natural infections appears less useful unless applied in conjunction with specific hybridization probes.