Introduction: Respiratory distress remains the most common cause of perinatal morbidity and mortality preterm infants. Ureaplasma urealyticum and U. parvum have been associated with respiratory diseases in premature newborns, but their role in the pathogenesis of the respiratory distress syndrome (RDS) is unclear. Aim of the Work: To investigate the colonization of the respiratory tract of preterm newborns by U. urealyticum U. parvum and Mycoplasma hominis by using Mycoplasma Duo kit and PCR to prove a possible association between the colonization of these microrganisms and the development of RDS with associated risk factors Also to compare the performance of the Mycoplasma Duo kit with PCR for the detection of Ureaplasma species. Subjects and Methods: Sixty paired endotracheal aspirates were obtained from premature infants ventilated due to hyaline membrane disease were analysed for detection of Ureaplasma spp. by Mycoplasma Duo assay and PCR. Results: This study revealed that the prevalance of Ureaplasma in neonatal endotracheal aspirates respiratory diseases was 33.3%, 35% as detected by Mycoplasma Duo kit and PCR tests respectively and U. parvum was found more than U. urealyticum in the study group. There was very good overall agreemenbetween the Mycoplasma Duo assay and PCR (0.96). As regarding to the average gestational age and birth weight there were a statistical significant difference between positive and negative case. Conclusion: Colonization of the lower respiratory tract by Ureaplasma spp. and particularly by U.parvum in preterm newborns was related to RDS. The Mycoplasma Duo assay is a commercially available is rapid and simple to use and has a sensitivity comparable to PCR for the detection of Ureaplasma spp. neonatal endotracheal aspirates. Also PCR could be a highly sensitive and specific technique for detecting Ureaplasma and for distinguishing U. urealyticum from U. parvum directly in clinical specimens.