Chromosomes of four species of Leishmania represented by ten different geographic isolates were analyzed by pulsed field gradient gel electrophoresis (PFG) to assess chromosome stability in these parasitic protozoans. Among different geographic isolates of the same subspecies, more than two-thirds of chromosomes had similar sizes, ethidium bromide staining intensities, and locations of alpha,beta-tubulin genes. However, among New World Leishmania, members of different species or subspecies have fewer than one-third of their chromosomes in common. Therefore, PFG karyotypes of Leishmania exhibit intraspecific variability similar to that reported for other parasitic protozoans. The greater similarities of the karyotypes of members of the same Leishmania subspecies may indicate that they represent valid taxa. These similarities also allowed the use of PFG in clinical diagnosis for rapid and accurate typing of patient isolates.