Brucellosis is a neglected bacterial zoonosis that has a significant impact on public health and the livestock industry. The purpose of this study was to determine the brucellosis seroprevalence, the circulating Brucella species, Brucella virulence-associated genes distribution, and repetitive extragenic palindromic PCR (REP-PCR) genotyping of Brucella isolates among ruminants in the Kafr Elsheikh Governorate. Blood samples were randomly collected from 926 cattle, 422 buffaloes, 301 sheep, and 333 goats in Kafr Elsheikh from January 2021 to December 2022. We determined apparent and true brucellosis prevalence in ruminants in various Kafr Elsheikh locations. Isolation and molecular confirmation (AMOS-PCR), phylogeny based on REP-PCR Brucella genotyping was inferred. Virutyping was performed on B. melitensis differences in virulence patterns. Brucellosis's true prevalence was calculated to be 13.3%, 11.2%, 15.8%, and 16.8% in cattle, buffaloes, sheep, and goats. Brucella isolates (n=8) were recovered from the supramammary lymph nodes and confirmed molecularly (AMOS-PCR) and bacteriologically as B. melitensis biovar 3. The B. melitensis isolates showed three virulence patterns (V1-V3), with V1 being the most common pattern (62.5%). REP-PCR clearly discriminated between the detected B. melitensis bv3 isolates into two clades (C1 and C2) and 7 REP genotypes (G1 – G7). In conclusion, the B. melitensis bv3 high diversity genotypes based on REP-PCR patterns highlight the cost-effective discrimination efficiency of the reproducible REP-PCR. B. melitensis's consistent isolation at low rates across the year of sampling may suggest various previous outbreaks of established infection rather than a recent introduction of foreign strains in the Governorate.