Background: Urinary tract infection is responsible for serious complications in pregnant women. Among different uropathogens, Ureaplasma urealyticum and Mycoplasma hominis are implicated in several urogenital infections. These organisms are not easily diagnosed by routine cultures and cannot be treated by common safe antibiotics used in pregnancy. This is the first study that aimed to rapidly detect these organisms in "urine of pregnant" women and determine their antibiotic susceptibility.Methods: A total of 1500 pregnant women were included, 750 symptomatic patients and 750 controls (apparently healthy). Urine samples were taken aseptically from all subjects and analyzed using the Mycoplasma System Plus (MSP) kit. The specimens were further subjected to multiplex polymerase chain reaction (PCR) for the molecular confirmation.Results: Of 750 urine specimens of the patients group, 223 specimens showed sterile pyuria suggesting the presence of mycoplasmas, whereas sterile pyuria was found in only 65 specimens of the control group. Of the 223 specimens of the patients group, 32 (14.3%) had U. urealyticum by MSP kit, 21 (9.4%) had M. hominis, 14 (6.3%) showed mixed coinfection, and 156 (70%) were negative. Seven (10.8%) of 65 specimens of the control group detected U. urealyticum. Conclusions:There was a good agreement between the MSP kit and the multiplex PCR. The MSP kit was an easy and rapid alternative to the complicated conventional culture method for the detection of urinary mycoplasmas. Doxycycline and minocycline had the highest sensitivity rates; however, clindamycin would be the first choice for empirical treatment of pregnant women.