This study was conducted to find incidence of Ureaplasma urealyticum (U. urealyticum) and Mycoplasma hominis (M. hominis) in 60 premature infants suffering (from chronic lung disease (CLD), and to evaluate the use of a prepared broth-agar culture system and Mycoplasma 1ST kit (bio-Merieux) for detection of these organisms. There was good agreement between the two systems for detection of the two organisms with sensitivity more than 76.9% and specificity more than 84.2%. Statistically there was no significant difference in the performance of the two systems (P>0.1) for both U. urealyticum and M hominis. Although the broth-agar culture system was considerably less expensive than the 1ST Mycoplasma kit, the Mycoplasma ST kit had the advantages of a long shelf life. The prevalence of colonization obtained for M. hominis and U. urealytium were high in the selected infants, being > 21.7% and > 41.7% respectively, and colonization of both organisms simultaneously was very common (40%). Conclusion: U. urealyticum and M. hominis must be considered in all cases of premature infants with CLD, and the Mycoplasma 1ST kit (bio-Merieux) provided more advantages than the prepared broth-agar system for detection of Mycoplasmas