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Objective This study assessed the antibiotic susceptibility and characterized antibiotic resistance genes of GBS isolates from selected tertiary care hospitals in Western province, Sri Lanka Methods A descriptive cross-sectional study was carried to determine antibiotic sensitivity of GBS among 175 pregnant women of >35 weeks of gestation attending antenatal clinics in four teaching hospitals. Low vaginal and rectal swabs were collected separately, GBS was identified by standard microbiological methods. Antibiotic sensitivity and minimum inhibitory concentration were done according to CLSI guidelines. DNA was extracted from culture isolates and resistance mechanisms were identified by PCR using ermB, ermTR, mefA and linB genes. Results GBS colonization in study sample was 25.7% (45/175) with detection rate of 22.9% and (40/175) and 2.9% (5/175) in vaginal and rectal samples respectively. All isolates were susceptible to penicillin with a MIC range of 0.03-0.12μg/ml. Seventeen (37.7%) were non-susceptible to erythromycin while 6 were intermediate and 11 were resistant. There were 15 (33.3%) non-susceptible isolates for clindamycin with 5 intermediate and ten resistant isolates. Of them seven had inducible clindamycin resistance (iMLSB). Erythromycin and clindamycin MICs ranged 0.03–0.32 μg/ml and 0.06–0.32 μg/ml respectively. The ermB gene was detected in 7(15.5%). The ermTR was found in 16 (35.6%) was significantly associated with iMLSB phenotype (P = 0.005). The mefA gene was detected in two (4.4%) isolates. The linB gene was not detected in tested isolates. Conclusion All isolates were sensitive to penicillin and the most prevalent resistance genotype was ermTR in the study population.