Tilapia (Oreochromis spp.) is globally used as an aquaculture fish species due to its high growth rate and disease resistance. However, it faces an increased risk of streptococcosis. Streptococcus agalactiae, also known as group B streptococcus (GBS), is the most important tilapia pathogen in Asia. Studies of Str.agalactiae infection in Taiwan are still unclear. Thus, this study aimed to explore the phenotype, genotype and pathogenicity of Str.agalactiae isolated from cultured tilapia in Taiwan in 2016-2018. The analysis revealed that 85% of the strains displayed β-haemolysis and 15% showed γ-haemolysis, with the same capsule level, and were positive for the CAMP test. The Rapid ID 32 Strep test showed a similarity of Rapid ID 32 Strep is more than 99.5% to GBS. Genotypic distribution by molecular serotyping detected only serotype Ia from all isolates, despite the regional differences. Pulsed-field gel electrophoresis (PFGE) was categorized into 3 and 10 clusters by restriction enzymes SmaI and ApaI, respectively. Virulence genes and antimicrobial resistance genes presented the same profile in all isolates. The challenge test with 106 CFU/fish (LD50 ), administered intraperitoneally, showed that the β-haemolysis strains had a higher mortality rate than γ-haemolysis, although they were from the same cluster from PFGE, year and region.