Objective To investigate the expression level of virulence-associated genes in promastigotes and amastigotes of different Leishmania spp.. Methods Total RNA was extracted from the promastigotes and amastigotes of Leishmania donovani, L. infantum, L. tropica, L. major and L. mexicana, and relevant strains. According to the reported gene sequences in GenBank, primers were designed in relation to the virulence-associated genes ［GDP-mannose pyrophosphorylase （GDPMP）, 3′a2rel-related protein （A2rel）, beta-galactofuranosyl transferase （LPG1）, lipophosphoglycan biosynthetic protein （LPG2）, kinetoplast membrane protein 11（KMP-11）, cpc gene for cysteine proteinase （CPC）, hydrophilic acylated surface protein （HASPB1）, cathepsin L-like cysteine protease （CPB2）, cathepsin L-like cysteine proteinase lmcpb2.8 （CPB2.8）, Mr 100 000 heat shock protein（CLP b）］, and control genes （alpha tubulin gene and GAPDH）. Semi-quantitative RT-PCR was performed to detect expression level of these genes in promastigotes and amastigotes of different Leishmania spp. Results There was a significant difference in the expression profiles of the genes among the promastigotes and amastigotes of different Leishmania spp. The HASPB1 was detected in the amastigotes of all strains and promastigotes of L. donovani, the GDPMP, LPG1, LPG2, CPB2.8, CPB2, CPC, A2rel and CLP b were expressed in the promastigotes and/or amastigotes of the specific Leishmania spp, respectively. None of the stains carried the KMP-11 gene, whereas the amastigotes of L. donovani SC10 strain and L. major 5ASKH strain possessed CPC. Conclusion The expression profile of the virulence-associated genes shows species-specific and stage-specific differences.