This article reports the development of a method for genotyping Chlamydia trachomatis, using PCR and sequencing of omp1, supplemented with three new variable number tandem repeat (VNTR) loci of C. trachomatis. Typeability, reproducibility and discriminatory power were assessed using four groups of samples: two groups (I and II) of C. trachomatis-positive patients and their positive partner(s), one group (III) of patients with recurrent or persistent C. trachomatis infections, and one group (IV) comprising samples containing a newly discovered mutant strain with a 377-bp deletion in the cryptic plasmid, the new variant C. trachomatis (nvCT). The VNTR loci (designated CT1335, CT1299, and CT1291) were all single nucleotide repeats chosen for maximal mutability and variation. In the study material, nine variants of CT1335, eight variants of CT1299 and five variants of CT1291 were found. The discriminatory power (D) of omp1 in the present material was D(omp1) = 0.69. Ds for VNTRs CT1335, CT1299 and CT1291 were 0.53, 0.74 and 0.74, respectively. The resolution power of the omp1-VNTR assay was 0.94. Stability over time of the VNTRs was investigated and found to be adequate for epidemiological studies. Using this genotyping assay, it was confirmed that the nvCT strain was indeed a clone. These results indicate that, with this novel method, strains of C. trachomatis can be individually identified, and epidemiological associations established.