BACKGROUND: Urinary tract infections (UTIs) rank among the most prevalent bacterial infections globally. Traditional urine culture methods have significant limitations in detection time and sensitivity, prompting the need to evaluate targeted next-generation sequencing (tNGS) as a potential diagnostic tool. METHODS: The study included a discovery cohort of 400 suspected UTI patients (202 analyzed) and a validation cohort of 200 patients (110 analyzed). The study assessed detection time, concordance rates, ability to identify polymicrobial infections, and antibiotic resistance genes (ARGs). Both clear and turbid urine samples were evaluated across different clinical settings. RESULTS: In the discovery cohort, tNGS demonstrated 96.5% concordance with culture-positive samples, while showing superior specificity in culture-negative specimens (53.1% vs 28.1% for mNGS). Detection time for tNGS (12.89 h) was notably shorter than mNGS (17.38 h) and traditional culture (61.48 h). tNGS exhibited remarkable capability in identifying polymicrobial infections (55.4% of samples), significantly outperforming both mNGS (27.7%) and traditional culture methods, which failed to detect any co-infections. The method showed particular strength in detecting fastidious organisms like Ureaplasma parvum and fungal species such as Candida tropicalis. For antibiotic resistance prediction, tNGS detected more ARGs (52.67% vs 41.22% for mNGS) and achieved 100% sensitivity for vancomycin and methicillin resistance in Gram-positive pathogens. The validation cohort confirmed tNGS's robust performance, maintaining high concordance rates for both culture-positive (90.00%) and culture-negative samples (55.00%), demonstrating consistent reliability across different clinical settings CONCLUSIONS: tNGS demonstrates advantages in rapid and accurate UTI diagnosis, particularly in detecting polymicrobial infections and analyzing antibiotic resistance genes. It shows promise as an effective complementary tool for UTI diagnostics.