Background: Mycoplasma hominis is often present in the amniotic cavity with microbial invasion associated with spontaneous preterm labor. Conventional culture method is the gold standard for detection of Mycoplasmas, but real-time polymerase chain reaction (real-time PCR) has revolutionized the diagnosis of M. hominis. Objective: The purpose of this study is the comparison of the culture methodology with real-time PCR for the detection of M. hominis in amniotic fluid samples. Methods: Amniotic fluid samples were collected from 65 pregnant women (age range: 25-45 years) previously followed at an infertility clinic. They were collected by transabdominal genetic amniocentesis during 16-21 weeks of gestation. Amniotic fluids were inoculated in SP4 broth for 48-72 h, and after becoming alkaline, culture suspension was spread on A7 agar plate for 1 week till the typical colonies seen in "fried-egg" morphology under stereomicroscope. DNA was extracted using a QIAGEN Mini DNA kit. The real-time-PCR was performed using Rotor-Gene Q Real-time PCR instrument. A melting-curve analysis was also performed. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were measured by real-time PCR by taking culture as gold standard. Results: Sixty-five women in 16-21 weeks of gestation, with a mean age of 33 +/- 5.06 years, were enrolled into this study. M. hominis detected by culture and real-time PCR assay was 72% (47/65) and 69% (45/65), respectively. 66% (43/65) specimens were positive by both methods. Real-time PCR sensitivity was 91.5%, specificity 88.9%, PPV 95.6%, and NPV 80%. Conclusion: Rapid detection of Mycoplasmas causing maternal complications such as neonatal infections and preterm labor in pregnancy by real-time PCR may be important and necessary. The high sensitivity and shorter time requirement of real-time PCR support its further development for diagnosis of Mycoplasma infections.