Sexually transmitted infections (STIs) are among the most common infections in Romania. Infection with Ureaplasma urelyticum is one of the major causes of STIs and can cause serious complications. Although tetracycline is the drug commonly used to treat infections caused by U. urealyticum, several studies indicate the emergence and rapid development of strains resistant to these antibiotics in the United States or Europe. Tetracycline resistance in bacteria is encoded by a number of different genetic determinants but in mycoplasmas the only tetracycline resistance determinant that has been reported is the tetM gene. Tetracycline resistance among Ureaplasma spp. is associated with the presence of the horizontally acquired tetM resistance gene. Our study on bacterial DNA aimed to determine the presence of tetracycline-resistant U. urealyticum strains, by identifying the presence of the tetM gene. We used first void urine samples from 622 STI-suspected subjects. DNA was extracted, purified and amplified via PCR for the simultaneous detection of 6 STIs. 68 patients were diagnosed with U. urealyticum. DNA obtained from these samples was amplified using the tetM gene and U. urealyticum - specific urease gene primers. The urease gene was amplified in all samples, confirming the presence of U. urealyticum. The tetM gene was amplified in 2 samples considered tetracycline-resistant strains. The study confirmed the presence of U. urealyticum strains resistant to tetracycline in Romania. The employed technique can produce quick results both for U. urealyticum detection and determination of its resistance to tetracycline using a single easy-to-collect biological sample.