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Background: This study was undertaken to determine the susceptibility profile and the mechanism of antibiotic resistance in Group B streptococcus (GBS) isolates detected in vaginal and rectal swabs from pregnant women attending Dr George Mukhari Academic Hospital, a University Teaching Hospital in Pretoria, South Africa. Methods: The samples were collected over an 11-month period, cultured on selective media (colistin and nalidixic acid agar and Todd-Hewitt broth), and GBS positively identified by using different morphological and biochemical tests. The susceptibility testing was done using the Kirby-Bauer and E test methods according to CLSI guidelines 2012. The D test method was used for the detection of inducible clindamycin resistance. Multiplex PCR with specific primers was used to detect different genes coding for resistance. Results: Out of 413 samples collected, 128 (30.9 %) were positive with GBS. The susceptibility testing revealed that 100 % of isolates were sensitive to penicillin, ampicillin, vancomycin and high level gentamicin. Erythromycin and clindamycin resistance was 21.1 and 17.2 %, respectively, in which 69 % had harboured constitutive macrolide, lincosamide and streptogramin B (MLSB), 17.4 % had inducible MLSB. The M and L phenotypes were present in 6.8 % each. The methylation of target encoded by ermB genes was the commonest mechanism of resistance observed in 55 % of isolates, 38 % of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was also distributed among the isolates. Conclusions: The study reaffirmed the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it identified the challenges of resistance to macrolides and lincosamides used as alternative drugs for individuals allergic to penicillin. More GBS treatment options for penicillin allergic patients need to be researched on. [ABSTRACT FROM AUTHOR]