ABSTRACT: Streptococcus agalactiae (also known Group B Streptococcus or GBS) represents the main pathogen responsible for early- and late-onset infections in newborns. The present study aimed to determine the antimicrobial susceptibility pattern and the capsular serotypes of GBS isolated in Eastern Sicily over 5 years, from January 2015 to December 2019. A total of 3494 GBS were isolated from vaginal swabs of pregnant women (37-39 weeks), as recommended by the Centers for Disease Control and Prevention. Capsular polysaccharide's typing of GBS was determined by a commercial latex agglutination test containing reagents to serotypes I-IX. The antimicrobial resistance pattern of GBS was determined through the disk diffusion method (Kirby-Bauer) and the double-disk diffusion test on Mueller-Hinton agar plates supplemented with 5% defibrinated sheep blood, according to the guidelines of the Clinical and Laboratory Standards Institute. Serotypes III (1218, 34.9%) and V (1069, 30.6%) were the prevalent colonizers, followed by not typable (570, 16.3%) and serotypes Ia (548, 15.7%), Ib (47, 1.3%), II (40, 1.1%), and IV (2, 0.1%). All 3494 clinical isolates were susceptible to cefditoren and vancomycin. Resistance to penicillin, ampicillin, levofloxacin, clindamycin, and erythromycin was observed in 6 (0.2%), 5 (0.1%), 161 (4.6%), 1090 (31.2%), and 1402 (40.1%) of the strains, respectively. Most of erythromycin-resistant GBS (1090/1402) showed the cMLSB phenotype, 276 the M phenotype, and 36 the iMLSB phenotype. Our findings revealed a higher prevalence of serotype III and a relevant resistance rate, among GBS strains, to the most frequently used antibiotics in antenatal screening.