Objective: To explore the correlations between group B Streptococcus (GBS) infection of the genital tract of pregnant women in the third trimester and premature rupture of membranes and neonatal outcomes. Methods: A total of 2 100 pregnant women in obstetric clinics from May 2017 to August 2019 were collected. Among them, 122 pregnant women with premature rupture of membranes in the third trimester were selected as an observation group, and 120 healthy pregnant women in the third trimester during the same period were selected as a healthy control group. GBS infection rates were compared between the 2 groups. According to whether the patients in the observation group were combined with GBS infection, they were divided into a GBS negative group and a GBS positive group. Single factor and multivariate logistic regression were used to analyze the risk factors of GBS infection in pregnant women with premature rupture of membranes, and to observe the adverse pregnancy conditions and neonatal outcomes in different groups. Results: The incidence of GBS infection in the healthy control group was lower than that in the observation group (P<0.05). There was a statistically significant difference in the incidence of abnormal fetal position, gemellary or multiple pregnancy and fetal macrosomia between the two groups (P<0.05). The results of multivariate logistic regression model analysis showed that abnormal fetal position, gemellary or multiple pregnancy and fetal macrosomia were the risk factors for GBS infection in pregnant women with premature rupture of membranes (P<0.05). The incidences of premature delivery, postpartum hemorrhage, and intrauterine infection in the GBS negative group were significantly lower than those in the GBS positive group (P<0.05). The incidences of neonatal asphyxia and neonatal pneumonia infection in the GBS positive group were higher than those in the GBS negative group (P<0.05). There was no significant difference in the incidence of neonatal infection between the 2 groups (P>0.05). Conclusion: Abnormal fetal position, gemellary or multiple pregnancy and fetal macrosomia can affect GBS infection in pregnant women with premature rupture of membranes in the third trimester. GBS infection can cause neonatal asphyxia, neonatal infection, and neonatal pneumonia, as well as adverse pregnancy outcomes such as premature birth, postpartum hemorrhage, and pneumonia infection, etc. In clinic, it is necessary to monitor and deal with the GBS infection of the genital tract of pregnant women in the third trimester in order to reduce the incidence of adverse maternal and infant outcomes.