Characterization of the kinetoplast DNA minicircles in a human pathogenic Indian isolate of Leishmania donovani has not been reported previously. Using inverse PCR, we constructed a library of PCR-amplified minicircle variable region from the kinetoplast DNA of this isolate. A combination of restriction enzyme digestion and nucleotide sequence analysis revealed five minicircle DNA sequence classes within the library, one of which was predominant, representing 75% of the kDNA network. Another distinct sequence class represented 15% of the minicircle network. Other minor sequence classes collectively constituted the remaining 10% of the network. Apart from generating basic information on the organisation and distribution of the different sequence classes within the minicircles, the DNA sequence analysis also revealed unique attributes to our minicircles. One was the surprising homology of our isolate (an Old World sp.) with distantly related New World Leishmania species. Secondly, open reading frames were also identified, indicating the possibility that these minicircles may have more than a structural role to play within the kinetoplast network.