Comparative genomic analysis of Vibrio harveyi, a leading pathogen in Mediterranean aquaculture, was conducted to assess the genomic plasticity of the species. V. harveyi is responsible for vibriosis outbreaks during the warmer months, resulting in significant economic losses that impact Greek aquaculture. Over a span of six years, we curated a diverse collection of bacterial strains associated with these outbreaks. Whole-genome sequencing was employed in 21 strains to uncover their evolutionary relationships and virulence factors. Pangenome analysis revealed significant gene gain/loss, with numerous unique genes within the strains. The core genome featured genes associated with pathogenicity, including secretion systems, flagella, pili, siderophores, and toxins. Furthermore, we examined the phenotypic traits and virulence of these strains using in vivo testing with gilthead seabream larvae. Our findings indicated variant metabolic profiles and virulence among the strains during these in vivo assays. By integrating genomic and phenotypic data, our study highlights the ongoing evolution of disease-associated V. harveyi strains, which pose a growing challenge to the aquaculture industry.