Aim: Chlamydia trachomatis (C. trachomatis) is an obligate intracellular pathogen which causes numerous diseases in both men and women, including sexually transmitted diseases. It is also an important neonatal pathogen, which can lead to infections of the eye and pulmonary complications. It is estimated that 75% of women and 50% of men with chlamydial infections are asymptomatic. Methods: This paper reports the results of a retrospective analysis of tests performed in the Department of Microbiology of Teaching Institute of Public Health in Rijeka during the year 2008. Overall 2804 genitourethral swabs from sexually active population between 13 and 65 years were examined. For routine diagnosis the following methods were used: direct immunofluorescence, enzyme-linked fluorescent assay – ELFA, and real time polymerase chain reaction – RT PCR. Results: C. trachomatis antigen was detected by enzyme-linked fluorescent assay in 2,9% of samples. Positive results were distributed almost equally among male (53%) and female (47%) patients, while age distribution analysis revealed that most of the positive patients belong to the age groups between 20 - 29 and 30 - 39 years. One hundred specimens admitted during December 2008 were tested with enzyme linked fluorescent assay and real time polymerase chain reaction. Enzyme linked fluorescent assay detected 8 positive specimens while polymerase chain reaction detected one additional positive specimen. Discussion and Conclusions: A reduction of the incidence of chlamydial infections is an important public health issue. The need for rapid, nonculture methods for the detection of C. trachomatis has led to the development of immunofluorescence assays, enzyme immunoassays, and nucleic acid amplification techniques - NAATs. Many studies have suggested that NAATs are considered to be important advance in the field of C. trachomatis detection and are now routinely used for C. trachomatis diagnosis.