We developed a 16S ribosomal (r) RNA gene-based PCR assay specific for Ureaplasma urealyticum and compared it with culture. We also wanted to assess the role of cervical U. urealyticum colonization in preterm births. Cervical swabs from 100 women with preterm contractions and from 50 asymptomatic pregnant women were collected and analyzed using PCR. The PCR and culture methods were compared using the samples from the asymptomatic patients. PCR and culture were equally effective at detecting U. urealyticum. Cervical colonization correlated with preterm delivery, with rates of 71% and 37% for those delivering preterm and those with term delivery, respectively (p = 0.008). The relative risk of preterm delivery if colonized with U. urealyticum was 3.34. 16S rRNA gene-based PCR proved to be a useful tool for detecting U. urealyticum compared to culture. Lower genital tract colonization with U. urealyticum was associated with preterm birth.