This study was aimed to identify and characterize Leishmania amastigote, and axenic form antigens. Two in vitro techniques were used to change leishmania parasite isolates from promastigote form to amastigotes and amastigote like (axenic) forms. The main strategy relied upon in vitro infection of murine macrophages cell line J774 with leishmania promastigote, at 37°C with 5% CO2, while the second technique relied upon the culture of promastigote at 37°C with low pH (5.5), and 5-10% CO2. Proteins were extracted and fractionated utilizing 12% Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE). Antigens were recognized using both immune dot blot and western blot procedures. PCR was performed for recognition of leishmania parasites in infected J774 macrophages. L. major was quicker in infectivity of macrophages cell line than L. donovani. Shared proteins ranging from 26-116 kDa were identified by SDS PAGE in all stages. Immune Dot-blot method showed positive outcomes, while western blot identified an exceptional antigen band of 16 kDa in amastigote, this unique band could be of value in diagnosis and vaccination of leishmaniasis. PCR results confirmed presence of both isolates demonstrating that coinfection is conceivable, and no indications of hereditary recombination at kinetoplast DNA (kDNA) were identified in macrophages simultaneously infected by L. major and L. donovani.