Background: Premature rupture of membranes (PROM) occurs 2.5–3 times more often in premature births than in births that are full term, is accompanied by many complications for both the mother and fetus and can cause serious adverse consequences. Little is known about the genetic susceptibility of PROM. Objective: The purpose of this study was to evaluate the association between several genotypes and PROM in a high-risk hospital-based population. Methods: The cases were 206 pregnant women who had PROM in labor, and the controls were 287 pregnant women who did not have PROM in labor. All subjects were selected between July 2006 and July 2007 from the Maternal and Child Health Hospital in Jinan City. We genotyped the following genes: the MTHFR C677T, MMP-9 C1562T, VDR C352T and IL-1β C3593T genotypes. The associations between the genotypes and risk of PROM were analyzed. Results: The CC genotype at MTHFR C677T, combined with bacterial vaginosis, was associated with a significantly increased risk of PROM (odds ratio, OR: 3.55; 95% confidence interval, CI: 1.05–12.02). Compared to those in the stratum of 40 or more weeks of gestation, those in the stratum of 37 or less weeks of gestation had an increased risk of PROM (OR: 5.11, 95% CI: 2.0–13.05 for the TT/TC genotype of MTHFR C677T; OR: 5.31, 95% CI: 1.07–26.44 for the TC/TT genotype of MMP-9 C1562T, and OR: 6.08, 95% CI: 1.39–26.60 for the TT genotype of VDR C352T). Conclusion: The results indicated that an interaction exists between genetic and environmental factors that are related to the occurrence of PROM.