Leptospirosis is recognized as the most widespread zoonosis with a global distribution. In this study, the antigenic variation in Leptospira interrogans and Leptospira borgpetersenii isolated from human urine and field rat kidney was preliminarily confirmed by microscopic agglutination test using monoclonal antibodies, and was further subjected to amplification and identification of outer membrane lipoproteins with structural gene variation. Sequence similarity analysis revealed that these protein sequences, namely OmpL1, LipL32 and LipL41, showed no more homologies to outer membrane lipoproteins of non-pathogenic Leptospira and other closely related Spirochetes, but showed a strong identity within L. interrogans, suggesting intra-specific phylogenetic lineages that might be originated from a common pathogenic leptospiral origin. Moreover, the ompL1 gene showed more antigenic variation than lipL32 and lipL41 due to less conservation in secondary structural evolution within closely related species. Phylogenetically, ompL1 and lipL41 of these strains gave a considerable proximity to L. weilii and L. santarosai. The ompL1 gene of L. interrogans clustered distinctly from other pathogenic and non-pathogenic leptospiral species. The diversity of ompL genes has been analyzed and it envisaged that sequence-specific variations at antigenic determinant sites would result in slow evolutionary changes along with new serovar origination within closely related species. Thus, a crucial work on effective recombinant vaccine development and engineered antibodies will hopefully meet to solve the therapeutic challenges. In this study, the antigenic variation in Leptospira interrogans and Leptospira borgpetersenii isolated from human urine and field rat kidney was preliminarily confirmed by microscopic agglutination test using monoclonal antibodies, and was further subjected to amplification and identification of outer membrane lipoproteins with structural gene variation. Sequence similarity analysis revealed that these protein sequences, namely OmpL1, LipL32 and LipL41, showed no more homologies to outer membrane lipoproteins of non-pathogenic Leptospira and other closely related Spirochetes, but showed a strong identity within L. interrogans, suggesting intra-specific phylogenetic lineages that might be originated from a common pathogenic leptospiral origin. Moreover, the ompL1 gene showed more antigenic variation than lipL32 and lipL41 due to less conservation in secondary structural evolution within closely rela The ompL1 gene of L. interrogans clustered distinctly from other pathogenic and non-pathogenic leptospiral species. The diversity of ompL genes has been analyzed and it envisaged that sequence-specific variations at antigenic determinant sites would result in slow evolutionary changes along with new serovar origination within closely related species. Thus, a crucial work on effective recombinant vaccine development and engineered antibodies will hopefully meet to solve the therapeutic challenges .