Leishmania major is the causative agent of cutaneous leishmaniasis. It is one of the most studied Leishmania species not only during vector interaction but also in the vertebrate host. Lipophosphoglycan (LPG) is the Leishmania multifunctional virulence factor during host-parasite interaction, whose polymorphisms are involved in the immunopathology of leishmaniasis. Although natural hybrids occur in nature, hybridization of L. major strains in the laboratory was successfully demonstrated. However, LPG expression in the hybrids remains unknown. LPGs from parental (Friedlin, Fn and Seidman, Sd) and hybrids (FnSd3, FnSd4A, FnSd4B, and FnSd6F) were extracted, purified, and their repeat units analyzed by immunoblotting and fluorophore-assisted carbohydrate electrophoresis. Parental strains have distinct profiles in LPG expression, and a mixed profile was observed for all hybrids. Variable levels of NO production by macrophages were detected after LPG exposure (parental and hybrids) and were strain specific.