Objective <p>To assess Group B <i>Streptococcus</i> (GBS) colonization, detection methods, and antibiotic susceptibility in late pregnancy and provide evidence to improve screening strategies and reduce perinatal infection risk.</p> Methods <p>This retrospective study included pregnant women screened for GBS between 2017 and 2024 at a tertiary hospital in Yunnan, China. Vaginal specimens were tested using culture or qPCR. Antibiotic susceptibility was evaluated for culture-positive isolates. Logistic regression was used to identify risk factors, and maternal and neonatal outcomes were compared between colonized and non-colonized women.</p> Results <p>The colonization rate was 3.9% among 5,184 women tested by culture and 7.3% among 6,303 women tested by qPCR. qPCR detection demonstrated significantly higher detection rates compared to culture-based methods in a paired comparison (<i>n</i> = 3242). All isolates were sensitive to penicillin G, vancomycin, and ampicillin, while high resistance was observed to clindamycin (73.7%), erythromycin (69.7%) and tetracycline (61.1%). Sensitivity to levofloxacin and moxifloxacin was 51.7%, with rare intermediate isolates. No resistance to linezolid, tigecycline, daptomycin, or vancomycin was detected. More than two miscarriages, gestational diabetes, and vaginitis were independent risk factors for GBS colonization. Colonized women had higher rates of premature rupture of membranes and chorioamnionitis, and their neonates had higher odds of pathological jaundice and fetal distress.</p> Conclusion <p>GBS colonization in this region was lower than national estimates, while qPCR offers markedly improved detection compared with culture. Local isolates show full susceptibility to penicillin, supporting its continued use as the first-line agent for intrapartum prophylaxis. Strengthened surveillance of colonization and resistance patterns, together with targeted prevention strategies, is warranted to reduce perinatal infection risk.</p>