We have examined the similarities and differences in the organization of tubulin genes in New World Leishmania by restriction endonuclease digestion of genomic DNA and Southern blot analysis, using heterologous and homologous tubulin gene probes. As judged by the hybridization pattern and the restriction fragment length polymorphism (RFLP), there were large differences in both the restriction and hybridization patterns of the β-tubulin sequences between stocks of the mexicana and braziliensis complexes. There were similarities in the hybridization patterns of different speciesof the mexicana complex. In contrast, a high heterogeneity was found between species of the braziliensis complex which includes intraspecific variation. The results suggest that this polymorphism may be associated with random mutations. The same analysis gave evidence of large differences in the β-tubulin gene restriction pattern between New and Old World Leishmania. This variation in the β-tubulin gene region was sufficient to distinguish between New and Old World Leishmania groups and between stocks of the mexicana and braziliensis complexes.