Background: Leishmania is a genus of trypanosomatid protozoa which causes a wide spectrum of diseases ranging from a self-healing cutaneous lesion to a vital visceral form of the disease. Although various experimental Leishmania vaccines have been prepared in different parts of the world, only a few of first generation vaccines have reached to human trials. Killed Leishmania major (KLM) and autoclaved Leishmania major (ALM) have been tested in human volunteers in Iran and other countries. We evaluated protein contents of KLM and ALM and fresh Leishmania major using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Methods: Leishmania major (MRHO/IR/75/ER) was isolated from infected BALB/c mice and cultured in Novy-MacNeal-Nicolle medium. It was then subcultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with fetal calf serum 20%. ALM and KLM were prepared by Razi Vaccine and Serum Institute of Iran. Electrophoresis of the two antigens and freshly cultured Leishmania major promastigotes was performed and their protein contents were compared using SDS-PAGE. Findings: The results of electrophoresis showed numerous bands from more than 100 kilodalton (kD) to less than 10 kD. KLM bands were found to be similar to freshly cultured intact Leishmania major (except for 57 and 24 kD bands). ALM contained two very close bands (71 kD) which were not seen in KLM or fresh Leishmania major. Conclusion: Identification and purification of protective immunogens in crude antigens and detection of their stability are essential in production of an effective vaccine. Keywords: Leishmania, Killed Leishmania major, autoclaved Leishmania major, Polyacrylamide gel electrophoresis