The molecular karyotypes of five species of Leishmania were studied by pulsed field gradient gel electrophoresis (PFGGE) of chromosome-sized DNA bands. Each species exhibits a unique pattern of 22-28 bands in the size range approximately 200-2200 kb whereas strains of one species exhibit similar karyotypes. Analysis of the behaviour of kinetoplast DNA during PFGGE showed that minicircle DNA remains confined to the gel slot but a proportion of the maxicircle DNA fractionates as a low molecular weight band below band 1. The band location of genes for alpha and beta tubulin, the 5' spliced leader sequence (5'SL), heat shock proteins 70 (hsp 70) and 83 (hsp 83) and thymidylate synthase-dihydrofolate reductase (TS-DHFR) were analysed. Housekeeping genes are not clustered in Leishmania but are found on at least 7 bands in L. major. The hsp 83 gene is linked to the tandemly repeated beta tubulin allele on band 21 in L. major. Among different species, the location of the unlinked hsp 83 and hsp 70 genes is conserved whereas the TS-DHFR and 5'SL sequences are found on bands of varying size. The 5'SL gene may be rearranged in L. enriettii and two 5'SL loci were identified in L. donovani and L. tropica. The conservation of loci in strains of L. major suggests that the chromosomal genetic linkage map should be a reliable marker for identifying unknown isolates of Leishmania. Sequences on one band in L. mexicana sp. were shared among several bands and distributed on homologous and non-homologous bands in other species showing that DNA sequences are rearranged during speciation in Leishmania.