Background. Maternal Streptococcus agalactiae (Group B Streptococcus (GBS)) colonization is an important cause of complications in mothers and neonates during gestation and after delivery. The data regarding GBS colonization among pregnant women in Palestine is scarce. The aim of this study is to determine the prevalence of GBS colonization, its associated risk factors, and the antibiotic sensitivity patterns in Nablus, West Bank, Palestine. Methods. A cross-sectional, single center study conducted at Rafidia Governmental Hospital in Nablus, West Bank, Palestine. Samples were collected between November 2019 and January 2020. Vaginal swabs from 200 pregnant women (≥35 weeks of gestation) attending the labor and delivery department were plated directly on CHROMagarTM StrepB (CHROM agar, France) and placed in an incubator at 35–37°C. After 24 and 48 hours, the plates were checked for growth and classified into three categories: growth of GBS with mauve colonies on chromogenic media, no growth, or other growth. The identification of the mauve colonies was confirmed by the CAMP test. Identified GBS isolates were tested for susceptibility to vancomycin, ampicillin, clindamycin, cefotaxime, erythromycin, and levofloxacin using the disc diffusion method. Clinical and demographic information were collected using a questionnaire. Result. The overall prevalence of GBS colonization was 12%. The median age of the study population was 27 years. GBS colonization was significantly associated with age ( p = 0.013 ), history of previous preterm delivery ( p = 0.013 ), and parity ( p = 0.015 ). No association was noted with smoking, previous abortion, previous history of fetal demise, vaginitis, or urinary tract infection. Resistance to ampicillin, vancomycin, cefotaxime, erythromycin, clindamycin, and levofloxacin was found to be 91.7%, 54.2%, 45.8%, 29.2%, 25%, and 8.3%, respectively. Conclusion. The prevalence of vaginal GBS in this study was 12% from Nablus, West Bank. Further research is needed to determine the GBS serotypes common in West Bank and the burden they cause on the health system. Moreover, this study also highlights the need to establish a screening program suited to a developing country with low control on the antibiotic’s prescription protocols.