The Chlamydia trachomatis major outer membrane protein gene (omp1) was amplified by polymerase chain reaction (PCR) from 89 clinical specimens, and restriction fragment length polymorphism (RFLP) patterns were analyzed after AluI or AluI and MspI digestion. Ten distinct RFLP omp1 genotypes were observed, although 4 types accounted for 70% of the isolates. The DNA sequence of variable domains (VDs) 1, 2, and 4 were determined for 49 samples. Thirty samples (61%) had prototype VD sequences, 15 (31%) had sequences that varied from prototype sequences, and 4 (8%) contained sequences from > 1 chlamydial isolates. Eleven of the 15 DNA sequence variations were one- or two-site nucleotide substitutions, 10 of which resulted in an amino acid change. Four DNA sequence variations were more extensive, with multiple nucleotide substitutions, insertions, or deletions. The data demonstrate that C. trachomatis isolates circulating in a relatively circumscribed geographic community exhibit remarkable omp1 DNA sequence polymorphism.