Different species of Leishmania are responsible for the diverse pathologies associated with leishmaniasis including Leishmania donovani which results in fatal visceral infection and Leishmania major which causes non-fatal cutaneous infection. In an attempt to identify genotypic differences between these related Old World Leishmania species which contribute to their distinct phenotypic characteristics, we have introduced a L. donovani cosmid library into L. major to select for L. donovani sequences which may increase L. major virulence in BALB/c mice. Through this approach, we have identified a region of the L. donovani genome which increased virulence in both visceral and cutaneous sites and was divergent from the corresponding region of the L. major genome. When these L. donovani sequences were reintroduced into L. major, they enhanced the overall virulence of L. major, increasing its ability to survive in both visceral and cutaneous sites. The region responsible for increased infection levels was determined to be the miniexon gene array derived from chromosome 36 of L. donovani. Pulse field electrophoresis revealed that L. donovani contained miniexon gene sequences in several chromosome locations as opposed to L. major which contains miniexon gene sequences only in chromosome 2. Because of the requirement for miniexon-derived transcripts in maturation of pre-mRNAs in trypanosomatids, this observation suggests that the increased expression of miniexon genes is associated with increased virulence. As the genome sequence for Leishmania becomes available, the in vivo selection procedure described within will be useful to identify additional species-specific sequences responsible for different pathogenic phenotypes associated with Leishmania infection.