In order to develop an indigenous and reliable immunodiagnostic assay for Chlamydia trachomatis in India, monoclonal antibodies (MAbs) were developed. Serovar D of C. trachomatis (which was previously isolated from the genital tract of infected women) was propagated, purified, and used for production of monoclonal antibody. A total of 12 murine hybrid clones producing immunoglobulin G (IgG) class monoclonal antibodies to C. trachomatis (species-specific, B serogroup-specific, and serovar-specific) were developed. Enzyme-linked immunosorbent assay (ELISA) was used to screen developed murine MAbs with C. trachomatis antigen. Dot-ELISA was used to check the specificity of clones and was used for selecting hybridomas that produced anti-C. trachomatis MAb. There was no cross-reactivity of species-specific, B serogroup-specific, and D serovar-specific anti-major outer membrane protein (MOMP) monoclonal antibodies with other species of Chlamydiae i.e., C. pneumoniae and C. psitacci. Immunoblotting was done for further characterization of six of these clones, i.e., B2.2 and D5.1 (B serogroup-specific), D10.4 and G1.5 (species-specific), and H5.6 and E4.2 (D serovar-specific). Three of these clones D10.4 (species-specific), B2.2 (B serogroup-specific), and H5.6 (D serovar-specific) which reacted with 40 kd MOMP protein in Immunoblotting were used for further screening to detect C. trachomatis in endocervical specimens. The percent positivity with these clones for detection of C. trachomatis antigen by enzyme immunoassay (EIA) was 45% with D10.4, 43% with H5.6, and 35% with B2.2, while 46% of the specimens were found positive by cell culture method. This indicates a high prevalence of C. trachomatis infection in the female genital tract. The sensitivity and specificity of developed anti- MOMP monoclonal antibody in EIA for chlamydial antigen detection was 91.3% and 94.4% for D10.4 clone (species-specific), 91.30% and 98.1% for H5.6 (D serovar-specific) and 75.00% and 99.07% for B2.2 (B serogroup-specific) compared to cell culture method.