Restriction endonuclease digestion of genomic DNAs from WHO Leishmania reference strains generates prominent kinetoplast DNA (kDNA) fragments ranging from 0.5–2.0 kb. Following fractionation by agarose gel electrophoresis, these DNA fragments were visualized as distinct bands in gels. Using as parameters the presence or absence of the prominent bands and their size and number, the Leishmania reference strains fell into three distinct genomic groups. These genomic groupings were applied to new Leishmania isolates. DNA analysis, involving restriction endonuclease digestion and Southern blot hybridization with probes generated from the prominent DNA bands of reference strains L. major IC-236 and L. major IC-235, cloned in plasmids, showed significant genetic variation within Leishmania and allowed some distinct isolates to be identified.