During studies in which we serotyped large numbers of Chlamydia trachomatis clinical isolates by using monoclonal antibodies, three novel serological variants (D-, D*, and I-) were identified. To determine the molecular basis for the altered monoclonal antibody reactions of these strains and other previously identified variants (Da, Ia, and L2a), we determined the nucleotide sequences of the variable domains in their major outer membrane protein genes. Da, D-, and D* variants differed by a single nucleotide and also an amino acid in the carboxy terminus of variable domain IV (VDIV) from the D serovar. The L2a variant also differed from L2 by a single amino acid but in VDII. Ia variants differed in VDI, III, and IV and I- variants differed in all four VDs from the I serovar. These studies demonstrate the potential for using major outer membrane protein VD sequencing as a highly sensitive typing method and further identify immunologically reactive major outer membrane protein epitopes.