Four polymerase chain reaction (PCR)-based approaches were used to analyze diversity within 23 Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single nonspecific primers, restriction analysis of the amplified ribosomal internal transcribed spacer (ITS) locus, single-stranded conformation polymorphism (SSCP), and sequencing of the ITS region. When PCR fingerprinting and restriction analysis of ITS were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave five different SSCP profiles among the 23 Sudanese isolates, whereas the ITS2 locus was highly conserved with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. SSCP analysis correlated well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigation of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics.