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Abstract: We have developed a high-resolution melting analysis (HRMA) for the genotyping of Chlamydia trachomatis and applied it specifically to the 11 sexually transmitted infection-related genotypes: D through K and L1 through L3. The variable segment 2 (VS2) was selected as the target for HRMA genotype identification. Eleven C. trachomatis genotypes were amplified by a nested real-time polymerase chain reaction (PCR) in the presence of the LCGreen saturating dye and showed no cross-reaction with 10 pathogenic bacteria or commensals from urogenital tract. The detection limit of HRMA method was 100 elementary bodies (EB)/mL. All of the 11 genotypes can be distinguished from each other by following an HRMA workflow. Genotype F, G, H, I, J, K, L2, and L3 could be directly identified from each other, whereas D, E, and L1 could be distinguished from each other by a second analysis with fewer curves or by heteroduplex formation with a known reference strain. In the validation panel of 36 C. trachomatis-positive urogenital samples genotyped by VS1–VS2 sequencing, nested real-time VS2 PCR followed by HRMA was able to discriminate between all samples correctly. This assay requires no fluorescence-labeled probes or separate post-PCR analysis and provides a simple and rapid approach for genotyping the C. trachomatis strains that are the most commonly sexually transmitted. [ABSTRACT FROM AUTHOR]