We have characterised 49 DNA probes specific for each of the six smallest chromosomes in Leishmania infantum and have examined the allocation of these probes in the molecular karyotypes of the other Old World Leishmania species Leishmania donovani, Leishmania major, Leishmania tropica and Leishmania aethiopica. These 49 probes define 6 physical linkage groups in the molecular karyotypes of various strains of L. infantum. 40 of these probes hybridise in the other Old World Leishmania species and show a remarkably conserved linkage pattern. No interchromosomal exchange nor fusion could be detected. Thus, in spite of the chromosomal size polymorphisms, the general structure of the genome seems to be conserved in the six smallest chromosomes among Old World Leishmania species. This structural genomic homogeneity should be helpful for mapping studies of any Old World Leishmania genomes.