The outcome of Leishmania infections is determined by both the parasite species and the host genetic makeup. While much has been learned regarding immune responses to this parasite, our knowledge on parasite-derived factors is limited. The recent completion of the L. major and L. infantum genome sequence projects and concurrent advancement in proteomics technology would greatly accelerate the search for novel Leishmania proteins. Using a proteomics-based approach to study species-specific Leishmania proteins, we developed high-resolution, broad pH (3-10) two-dimensional gel electrophoresis (2-DE) separations to determine protein-expression profiles between highly infectious forms of the parasitic species L. amazonensis (New World) and L. major (Old World). Approximately 1,650 and 1,530 distinct protein spots were detected in the L. amazonensis and L. major gels, respectively. While a vast majority of the spots had similar distribution and intensity, a few were computationally defined as preferentially expressed in L. amazonensis in comparison to L. major, or vice versa. These data attest to the feasibility of establishing a 2-DE-based protein array for inter-species profiling of Leishmania proteins and provide the framework for future design of proteome studies of Leishmania.