Aim. Polymerase chain reaction (PCR) is a key method for the C. trachomatis diagnostics. The first-generation tests targeting a cryptic plasmid showed quite a high sensitivity; however their value has recently been compromised by the discovery of C. trachomatis strains lacking the target DNA segment (e.g. the “Swedish” variant) and thus escaping the diagnostics. Moreover, there are variants bearing no plasmid at all. We propose the addition of a chromosome gene as a PCR tar-get to back up plasmid-based assays and enhance the overall efficiency of diagnostics. Methods. Two multiplexed PCRs were set up to target C. trachomatis cryptic plasmid and the 16s rRNA gene. The 16s rRNA primers produce PCR signal from a range of Chlamydia species whereas the introduction of a Taqman probe (essential for real-time PCR) scales the assay down to C. tra-chomatis. At the same time, our plasmid PCR is specific to C. trachomatis exclusively. Results. The sensitivity of plasmid and 16s rRNA PCRs reached from one to ten genome-equivalents per reaction (geq/rxn) whereas the efficiency was always about 100%. Multiplexing did not reduce the analytical sensitivity. Addition of DNA prepared from clinical specimens to the reaction mix did not affect PCR with pure C. trachomatis DNA further demonstrating the robustness of this system. The kinetics of the two reactions was compared in 49 DNA samples prepared from C. trachomatis-positive swabs. In 45 of these samples, the reactions showed a good correlation in the threshold cycle of amplification Cq, the main analytical parameter of real-time PCR. Conclusions. The simultaneous detection of chromosomal and plasmid targets in multiplex PCR offers a high sensitivity and is particularly advantageous for specimens where the plasmid might be lost due to DNA degradation or counter-selection after treatment. The dual strategy of PCR presented here could constitute the core of a diagnostic test for both in-house and commercial use.