Background: Streptococcus agalctiae (Group B Streptococcus, GBS) is a perinatal pathogen and a leading cause of neonatal infections worldwide. Serotype, sequence type, clonality, antibiotic resistance genes and surface protein profiles of GBS are scarce in Ethiopia, a reason that this study was planned to investigate. . Methods: Sixteen colonizing GBS isolates obtained from recto-vaginal swabs of pregnant women and body surfaces of newborns were further analyzed. Minimum inhibitory concentration (MIC) test, and whole genome sequence (WGS) methods were done for antibiotic susceptibility test, and molecular characterization of the isolates. Results: All the GBS isolates analyzed were belonged to four capsular serotypes: II, 11/16(68.8%), V, 3/16(18.8%), Ia and VI each with 1/16(6.3%) and five sequence type (ST-2, ST-10, ST-14, ST-569 and ST-933). Sequence type-10 was the most predominant ST followed by ST-569. The five STs were grouped into the four clonal complexes (CC -1, CC-10, CC-19, and CC-23). Different surface proteins and pili families such as ALP1, ALPHA, ALP23, PI-1 / PI-2A1, PI-1 / PI-2B, and Srr1 were detected from WGS data. All isolates were found to be susceptible to the tested antibiotics except for tetracycline in MIC and WGS test methods used. Tetracycline resistant determinant genes such as TETM and TETL / TETM combination were identified. Conclusion: Further studies on serotype and molecular epidemiology will provide a comprehensive data of the GBS capsular serotype and clones available in Ethiopia.