Chlamydia trachomatis (C. trachomatis) is a leading cause of bacterial sexually transmitted infections in developed and undeveloped countries, and therefore a global public health issue. In an era of increasing bacterial resistance to antibiotics, resistance has been an exceedingly rare phenomenon in C. trachomatis; however, clinical treatment failures attributed to multidrug-resistant C. trachomatis strains have been described on several occasions. Cell culture systems using McCoy cells and subsequent immunofluorescent staining are still the most common methodology used for antimicrobial susceptibility testing, but the presence of resistance markers should be appraised by further genetic analysis. Azithromycin resistance of C. trachomatis is often a result of the mutations in the peptidyl transferase region of 23S rRNA genes, tetracycline resistance is usually linked to the presence of foreign genomic islands integrated in chlamydial chromosome, whereas a predominant mechanism of fluoroquinolone resistance is a point mutation in the gyrA quinolone-resistance-determining region. A nucleotide substitution in rpoB gene is responsible for rifampin resistance, and different mechanisms have been involved in the development of resistance to aminoglycosides, lincomycin and sulphonamide/trimethoprim combinations.