In an analytical methods comparison study on clinical samples, the Abbott RealTime CT new formulation assay (m2000 real-time PCR) consisting of a duplex PCR targeting different parts of the cryptic plasmid in Chlamydia trachomatis was compared with version 2 of the Roche COBAS(R) TaqMan(R) CT assay comprising a duplex PCR for a target in the cryptic plasmid and the omp1 gene, and compared with the Gen-Probe APTIMA COMBO 2(R) assay (AC2) targeting the C. trachomatis 23S rRNA molecule. First-catch urine samples from Sweden were tested in Malmoe for C. trachomatis with the m2000 real-time PCR assay, and with an in-house PCR for the new variant C. trachomatis strain with a deletion in the cryptic plasmid. Aliquots of the urine samples were sent to Aarhus, Denmark and further examined with the TaqMan CT and the AC2 assay. A positive prevalence of 9.1% (148/1,632 urine samples examined) was detected according to the combined reference standard. The sensitivity and specificity of the three assays were as follows: for Abbott m2000, 95.3% (141/148) and 99.9% (1,483/1,485), respectively; for Roche TaqMan, 82.4% (122/148) and 100.0% (1,485/1,485); for Gen-Probe AC2, 99.3% (147/148) and 99.9% (1,484/1,485). The plasmid mutant strain was detected in 24% (36/148) of the C. trachomatis positive samples. There is a difference in sensitivity between the new formulation of the Abbott and the Roche assays, but both assays detected the wild-type and the new variant C. trachomatis strains equally well.