Restriction analysis of total DNAs from Leishmania donovani complex strains revealed heterogeneous distribution of kDNA fragments among the reference strains of L. donovani. The cloned kDNA fragments used as probes in the hybridisation analysis have proven useful for identifying and characterising L. donovani subspecies when screening newly isolated leishmanial parasites from patients, vectors and reservoir hosts. This genetic heterogeneity was confirmed by karyotyping by pulsed field gel electrophoresis of four L. donovani reference strains. A high degree of polymorphism was shown, affecting not only the chromosome size, but also their number.