To investigate the group B streptococcus (GBS) infection status in genital tract of pregnant women during the third trimester of pregnancy, and to study its influence on the premature rupture of membranes and the pregnancy outcomes of the women. Methods: 3,970 pregnant women during the third trimester of pregnancy who received regular prenatal examinations were given GBS infection detection from June 2021 to July 2023. These women were divided into group A (women with GBS infection) and group B (women without GBS infection). The rates of the premature rupture of membranes and the adverse pregnancy outcomes of the women were compared between the two groups. Results: Among 3,970 pregnant women, 103 women were infected GBS in group A, with an infection rate of 2.6%. Another 103 women who had been randomly selected from the women without GBS infection were included in group B. In group A, the infection rate of the women ≤30 years old (68.0%) was significantly higher than that of the women with 30-35 years old and that of the women >35 years old. The incidence of the premature rupture of membranes (18.5%) of the women in group A was significantly higher than that (4.9%) of the women in group B. The incidences of chorioamnionitis (6.8%), the neonatal pneumonia (13.6%), the fetal growth restriction (8.7%), the neonatal infection (7.8%), the fetal intrauterine distress (9.7%) of the women in group A were significantly higher than those (0, 4.9%, 1.0%, 1.0% and 1.9%) of the women in group B (all P<0.05). There were no significant differences in the incidences of the maternal fever during labor and the neonatal jaundice between the two groups (P>0.05). Conclusion: The GBS infection in genital tract of the pregnant women during the third trimester of pregnancy will increase the incidence of premature rupture of membranes and the risk of adverse pregnancy outcomes. Therefore, the clinical intervention measures should be taken for the women with GBS infection in time.