Mycoplasma hominis one of the smallest free-living prokaryotes that lack peptidoglycan in the cell wall. It is also a well-known pathogenic Mycoplasmain the human reproductive system that causes abortion, bacterial vaginosis, infertility, and a low birth weight. This research investigated the incidence of preterm rupture of membrane (PROM)inpregnant women due to M.hominis in the Alavi Hospital of Ardabil Province using twocomparative PCR techniques.This study evaluated diagnostic tests on 100 pregnant womenwith rupturedmembraneand 20 healthy women withno symptoms (control) referred to the Alavi Hospital in Ardabil Province. After sampling, the samples were transferredto the laboratoryin tubes containing PBS and PPLO.Using culture ofsamples from 100 pregnant women with PROM, 41 samples (41%) were positive for the presence of M.hominiswhereas PCR revealedpositive results in62 samples (62%). PCR method displayed positive casesin 2 (10%) out of 20patients in the control group. PCR results were not significantly correlatedwith education level, parity, and gestational age. A direct correlationwas observed between the agesof pregnant women with vaginal infections before pregnancy, history of abortion, and in women with PROM.The incidence of PROMin pregnant women caused by infection of the bacterium M.hominis is high. PCR technique is more appropriatecompared with the culture method to detectM.hominis positive cases in pregnant women with PROM.