Genotyping of Chlamydia trachomatis is limited by the low sequence variation in the genome, and no adequate method is available for analysis of the spread of chlamydial infections in the community. We have developed a multilocus sequence typing (MLST) system based on five target regions and compared it with analysis of ompA, the single gene most extensively used for genotyping. Sequence determination of 16 reference strains, comprising all major serotypes, serotypes A to L3, showed that the number of genetic variants in the five separate target regions ranged from 8 to 16. The genetic variation in 47 clinical C. trachomatis isolates of representative serotypes (14 serotype D, 12 serotype E, 11 serotype G, and 10 serotype K strains) was analyzed; and the MLST system detected 32 variants, whereas 12 variants were detected by using ompA analysis. Specimens of the predominant serotype, serotype E, were differentiated into seven genotypes by MLST but into only two by ompA analysis. The MLST system was applied to C. trachomatis specimens from a population of men who have sex with men and was able to differentiate 10 specimens of one predominant ompA genotype G variant into four distinct MLST variants. To conclude, our MLST system can be used to discriminate C. trachomatis strains and can be applied to high-resolution molecular epidemiology.Chlamydia trachomatis can be subdivided by serological typing, based on the major outer membrane protein, into at least 15 serotypes. Serotypes A to C are associated with ocular trachoma, serotypes D to K preferably colonize the urogenital tract, and serotypes L1 to L3 cause lymphogranuloma venereum.Serotyping of Chlamydia is laborious since it requires multiple passages in cell culture and the use of a large panel of monoclonal antibodies. Genotyping methods are more commonly used, and the major outer membrane protein gene, ompA, provides the best discriminatory capacity of the genes tested. Restriction fragment length polymorphism analysis of ompA is rapid, and its results show a high level of agreement with the results serotyping (9), while DNA sequencing of ompA has a higher resolution and can discriminate strains in clinically high-risk populations (2, 14). However, when it is applied to nonselected populations, the limited resolution of ompA sequencing restricts the amount of epidemiological information that can be obtained (6). This is especially true, given that the single serotype E comprises almost half of all urogenital chlamydial infections, and within this serotype, one genotypic variant appears to predominate (3, 4, 6). There is therefore an obvious need to develop better methods for evaluation of the molecular epidemiology of chlamydial infections. Furthermore, it has recently been shown that mutant strains evade systems commonly used for the detection of C. trachomatis (11). At present little is known about the spread of such changed chlamydial strains, but they are known to be prevalent in several regions of Sweden and are probably prevalent elsewhere. In this conte...