Streptococcus iniae causes high mortalities in aquaculture farms all over the world. This study aimed to isolate, identify pathogenicity and genetic relatedness of S. iniae obtained from cultured Nile tilapia (O. niloticus) at Kafr El-Shiekh Governorate. Fourteen isolates of pure S. iniae cultures were biochemically identified, and confirmed through amplification and sequencing of the 16S rRNA gene. Experimental challenge with six selected isolates resulted in marked clinical signs similar to those recorded in natural infections. The LD₅₀ values were ranged between 3.7×10⁷ to 4.3×10⁸ cfu/fish. All strains were sensitive to oxytetracycline and florfenicol. In repetitive sequence-based PCR (rep-PCR) analysis using the BoxA primer, isolates presented genetic heterogeneity and were divided into six genotypes banding patterns. Understanding the epizootiology and genetic variability between S. iniae isolates, could help for better control and vaccine development.