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Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. As reported in other countries, after the rise and fall of the pandemic strain in Chile, other post-pandemic strains have been associated with clinical cases, including strains lacking the major toxins TDH and TRH. Since the presence or absence oftdhandtrhgenes has been used for diagnostic purposes and as a proxy of the virulence ofV. parahaemolyticusisolates, the understanding of virulence inV. parahaemolyticusstrains lacking toxins is essential to detect these strains present in water and marine products to avoid possible food-borne infection. In this study, we characterized the genome of four environmental and two clinical non-toxigenic strains (tdh-,trh-, and T3SS2-). Using whole-genome sequencing, phylogenetic, and comparative genome analysis, we identified the core and pan-genome ofV. parahaemolyticusof strains of southern Chile. The phylogenetic tree based on the core genome showed low genetic diversity but the analysis of the pan-genome revealed that all strains harbored genomic islands carrying diverse virulence and fitness factors or prophage-like elements that encode toxins like Zot and RTX. Interestingly, the three strains carrying Zot-like toxin have a different sequence, although the alignment showed some conserved areas with thezotsequence found inV. cholerae. In addition, we identified an unexpected diversity in the genetic architecture of the T3SS1 gene cluster and the presence of the T3SS2 gene cluster in a non-pandemic environmental strain. Our study sheds light on the diversity ofV. parahaemolyticusstrains from the southern Pacific which increases our current knowledge regarding the global diversity of this organism