Yersinia ruckeriis a highly virulent Gram-negative fish pathogen affecting many farmed and wild fish species. In the present study, Y. ruckerihas been isolated from 6 tilapia (Oreochromis niloticus) farms located in Kafrelsheikh Governorate. Studied farms suffered from increased fish mortality just after the decreased feed intake; the most common clinical signs were the erythematic appearance of lips, congestion to extensive haemorrhages all over the external body surface, and fins and tail erosions. Internally, diseased fishes showed petechial haemorrhages on the external hepatopancreas surface with engorged gall bladder filled with faint-coloured bile. The spleen and posterior kidney were congested, and the haemorrhagic gastroenteritis was also dominant. Out of 150 clinically diseased fish, 96 Y. Rucker were biochemically identified, and 45 were isolated from the liver. Molecular screening of selected isolates for certain virulence genes indicated that Yrp < /em>1 was the most common virulence gene present in 60% of tested isolates, followed by YhlA gene in 40 %, and finally, Yrlnv gene in 20%, while YhlB gene was completely absent. All the tested Y. ruckeri isolates were virulent for O. niloticus; challenge test indicated the presence of a direct relation between isolate pathogenicity and isolate virulence genes content (genotype). Isolate with 2 virulence genes was more pathogenic than that with one or without any virulence gene. The LD₅₀ ranged between 3.9x10⁶ and 9.7x10⁶ for the studied isolates. In conclusion, Y. ruckeri was responsible for the disease condition affecting the studied Nile tilapia farms.