Protozoan parasites of the Leishmania donovani complex (L. donovani, L. infantum/L. chagasi) are causative agents of visceral leishmaniasis. To understand phylogeny and taxonomy within this group better we have developed 2 new polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) analyses of the major surface protease (msp or gp63) intergenic (ITG) regions. We have named this approach msp intergenic region RFLP typing (MIRT). One intergenic region lies between the constitutive msp (mspC) and stationary phase msp (mspS4) genes (ITG/CS) and the other between multicopy logarithmic phase msp (mspL) genes (ITG/L). The markers generated robust and congruent phylogenies, identifying 5 genetic clusters within L. donovani. One cluster was synonymous with L. infantum (L. chagasi); clusters strongly correlated with isoenzyme typing and some with geographical origin. These genetic groups may be important for epidemiological and clinical studies. The congruence of the groups identified indicates suitability of these genomic targets for taxonomic studies. Furthermore, subgroups of L. donovani were of equivalent phylogenetic status to L. infantum. No evidence was found to support the existence of L. archibaldi. It is likely to be necessary in future to re-evaluate the taxonomic status of L. donovani or of L. infantum, as discrete species.