Ureaplasma parvum and Ureaplasma urealyticum can colonize the urinary and genital tracts and cause various infections. Due to their fastidious growth requirements, these organisms cannot be identified by routine laboratory diagnostic methods. Therefore, more detailed studies are needed to investigate their incidence in the community, resistance characteristics, and clinical manifestations. This study aimed to demonstrate the presence of ureaplasmas in patients with suspected urinary tract infections. As a part of the study objectives, a commercial real-time quantitative polymerase chain reaction (qRt-PCR) panel kit targeting U.urealyticum and U.parvum was used and the results were confirmed by sequence analysis. A total of 603 midstream urine samples submitted to our laboratory from patients with suspected urinary tract infections were analyzed using a urinary tract infection qRt-PCR panel kit (Bioeksen, Türkiye). Of the 195 samples found positive for U.urealyticum and/or U.parvum, 130 with sufficient sample volume were subjected to next generation sequencing (NGS) using the MinION™ system with a rapid barcoding kit (SQK-RBK110.96), both from Oxford Nanopore Technologies® (UK), according to the manufacturer’s instructions after storage at -20 °C. Of the 195 (32.33%) samples found positive by qRt-PCR, 138 (70.76%) were positive for U.parvum, 46 (23.58%) for U.urealyticum and 11 (5.64%) showed the presence of both species. Sequence analysis was performed on 130 of the 195 qPCR-positive samples that had sufficient volume. In 17 of these samples, no amplification product was observed during electrophoresis and five yielded insufficient sequencing data (< 10 coverage). As a result, sequence analysis data could not be obtained for 22 samples, while valid results were obtained for 108 samples. Among these 108 samples, 102 (94.44%) showed concordant results between qPCR and NGS, while six (5.56%) showed discordant results. One of the six discordant samples was identified as U.urealyticum by qPCR, while it was identified as U.parvum in the sequence analysis. In the remaining five samples, only one of the two species detected by qPCR could be demonstrated by NGS, the other could not be demonstrated. In three of the five samples, only U.parvum could be demonstrated by sequence analysis and in two, only U.urealyticum could be demonstrated. In the study, U.parvum and/or U.urealyticum positivity was found at a rate of 32.33% in midstream urine samples. In this study, it was aimed to draw attention to the fact that these microorganisms may be a possible cause of urinary tract infections due to their high positivity rates and they should not be ignored in clinical evaluations.