Background: Growth of Mycoplasma in genital tract can cause problems such as infertility, pelvic inflammatory disease (PID), and preterm labor. This study was designed to evaluate the role of these bacteria in preterm labor among individuals in Gorgan city which is located in north of Iran. Methods: The study included 100 women with complaints of pain in preterm labor before 37 weeks of pregnancy (case group) and 100 women with term labor (control group) who were referred to Shahid Sayyad Shirazi Teaching Hospital in Gorgan city, north of Iran. Vaginal swabs, collected from all of these women, were evaluated for genital Mycoplasma sp. by molecular method using specific primers with poly-merization chain reaction (PCR). The comparison of results was done by conducting X 2 and p<0.05 was considered significant. Results: Genital Mycoplasma was detected in 78 cases (39%) of 200 vaginal samples. Genital Mycoplasma colonization rates in the preterm and term samples were 60% and 18%, respectively, with relative risk of 2.05 (1.78-2.37) (p=0.001). The proportion of Ureaplasma parvum (44% and 15%), Ureaplasma urealyticum (11%, 3%), and Mycoplasma homins (5%, 0%) was significantly higher in women with preterm birth (PTB) than term labor. No cases of Mycoplasma genitalum were detected in this study. Conclusion: There is a significant relationship between presence of genital Mycoplasma in vaginal secretion and the risk of preterm labor.