Humans and marine mammals such as sea otters (Enhydra lutris) are exposed to pathogenic Vibrio species through consumption of marine invertebrates and contact with coastal waters. Despite the disease threats of vibriosis in humans and marine mammals, One Health focused Vibrio spp. comparative epidemiology studies are limited. This dissertation combines often-disparate approaches of comparative genomics and epidemiology to advance research for vibriosis management in three aspects: 1) antimicrobial resistance, 2) virulence detection, and 3) diagnostic resolution.Chapter 1 characterized both phenotypic (n=165) and genomic (n=444) antimicrobial resistance patterns of Vibrio spp. isolated between 2000-2019 from coastal Alaska, California, and Washington and from various sources including southern sea otters (Enhydra lutris nereis), northern sea otters (Enhydra lutris kenyoni), shellfish, and environmental samples. Using genomic methods, a previously misidentified species, Vibrio diabolicus, was detected in northern and southern sea otters and their coastal environment. Ampicillin resistance was common in Vibrio spp. although there was discordance between genotype and phenotype best exemplified in V. parahaemolyticus isolates. Discordance was partially explained by allelic variation in the beta-lactamase gene blaCARB. This study provides useful insights into antimicrobial treatment of vibriosis in sea otters and identifies genotypes associated with ampicillin resistance. Chapter 2 characterized genetic markers of virulence in Vibrio spp. across isolation sources using the Chapter 1 genomic dataset (n=444) along with public domain V. parahaemolyticus genomes (n=126) isolated from humans and shellfish in North America. Vibrio parahaemolyticus (n=287) isolated from humans and sea otters were more likely to carry virulence factors such as hemolysins and secretion system genes than isolates from environmental sources. Despite general genomic diversity of V. parahaemolyticus, genome clusters with high similarity were detected which included virulence factor positive genomes from both humans and sea otters. V. parahaemolyticus strains with co-occurrence of two virulence-related secretion systems were more likely to be isolated from dead southern sea otters with septicemia, enteritis, and moderate to severe melena than V. parahaemolyticus strains without either secretion system. This study provides evidence that V. parahaemolyticus undergoes selection pressures in both humans and otters resulting in apparent expansion of virulent strains and provides insights into V. parahaemolyticus pathogenicity in sea otters. Chapter 3 assessed the diagnostic resolution of closely related species in the Harveyi clade by combining the V. alginolyticus, V. diabolicus, and V. parahaemolyticus genomes from Chapter 2 with 150 additional genomes from the public domain. Within public domain genomes, 28 V. diabolicus were incorrectly labeled as V. alginolyticus resulting in 10 V. diabolicus genomes from human cases. A pangenome-wide association study identified species-selected gene clusters as well as V. alginolyticus and V. diabolicus virulence-related genes associated with either humans or sea otter isolates. Novel associations between putative virulence genes or alleles and either human or sea otter hosts were identified that were not present in virulence factor databases. These findings suggest V. diabolicus is an overlooked pathogen of humans and sea otters due to its close phylogeny with V. alginolyticus and provides additional diagnostic information to better differentiate clinical strains of each species in otters and humans. This research utilized genomic data for comparative epidemiological investigations of Vibrio spp. in both northern and southern sea otters and presents evidence that sea otters are at risk for antimicrobial resistant and virulent Vibrio strains, including the first documentation of V. diabolicus in sea otters. These chapters provide a baseline for further surveillance and include insights to improve the diagnosis and treatment of pathogenic Vibrio spp. in sea otters. Additional spatiotemporally linked genomic surveillance of Vibrio spp. in sea otters and humans is needed, which could further elucidate the potential for shared transmission routes of virulent and antimicrobial resistant vibriosis.