Klebsiella pneumoniae is a major human pathogen and a significant cause of nosocomial infections. Multiplex PCR proved to be a precise, rapid, and cost-effective method for detecting the presence of the causative pathogen in a significantly shorter time frame compared to conventional culture methods. The detection and identification of pathogens using molecular methods facilitate rapid and accurate diagnosis. The aim of the present study was to rapidly and directly identify nalidixic acid-resistant Klebsiella pneumoniae in urine samples using the multiplex polymerase chain reaction (PCR) method, in comparison with conventional culture techniques. A total of 230 urine samples were randomly collected from Ghaem Hospital in Mashhad. To detect the presence of Klebsiella pneumoniae and genes associated with nalidixic acid resistance, bacterial DNA was directly extracted from the urine sediment. The extracted DNA was analyzed for the presence of the 16S rRNA gene and resistance-related genes, namely qnrA, qnrB, qnrS, and aac(6’)-Ib-cr, using the multiplex PCR method. For validation and comparison, phenotypic testing was also performed, including urine culture and antibiotic susceptibility testing using the disk diffusion method. Among the 230 urine samples, Klebsiella pneumoniae was identified in 17.39% of cases using direct multiplex PCR. The three genes Kp/16S rRNA, qnrB, and qnrS were simultaneously present in 50% of the urinary samples analyzed. Resistance to nalidixic acid was observed in 42.5% of the isolates, while resistance to norfloxacin, ciprofloxacin, and ofloxacin was found in 40% of the isolates. Multiplex polymerase chain reaction has high sensitivity and accuracy due to the use of multiple primer pairs simultaneously. On the other hand, the application of this technique directly on clinical samples will help in the rapid identification of infectious agent isolates and antibiotic-resistant strains.