A method for detection and genotyping of genital Chlamydia trachomatis infections based on omp1 gene amplification and sequencing was developed. DNA was extracted from urogenital or urine samples using a Chelex-based method, and an approximately 1,100-bp-long fragment from the omp1 gene was directly amplified and sequenced. Genotyping was performed by BLAST similarity search, and phylogenetic tree analysis was used to illustrate the evolutionary relationships between clinical isolates and reference strains. The method was used to determine the genotypes of C. trachomatis in 237 positive urogenital and/or urine specimens collected at a Swedish sexually transmitted disease clinic during 1 year. The most common genotypes corresponded to serotypes E (47%) and F (17%). The omp1 gene was highly conserved for genotype E (106 of 112 samples without any mutation) and F (41 of 42 samples without any mutation) strains but appear slightly less conserved for genotypes G (n = 6) and H (n = 6), where the sequences displayed one to four nucleotide substitutions relative to the reference sequence. Genotyping of samples collected at the follow-up visit indicated that two patients had become reinfected, while three other patients suffered treatment failure or reinfection. One woman appeared to have a mixed infection with two different C. trachomatis strains. This omp1 genotyping method had a high reproducibility and could be used for epidemiological characterization of sexually transmitted Chlamydia infections.