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<i>E. coli</i> was isolated from the Salish Sea (Puget Sound) ecosystem, including samples of marine and fresh water, and wildlife dependent on this environment. <i>E. coli</i> isolates were assessed for phenotypic and genotypic resistance to antibiotics. A total of 305 <i>E. coli</i> isolates was characterized from samples collected from: marine water obtained in four quadrants of the Salish Sea; select locations near beaches; fresh water from streams near marine beaches; and fecal samples from harbor porpoises (<i>Phocoena phocoena</i>), harbor seals (<i>Phoca vitulina</i>), river otters (<i>Lontra canadensis</i>), and English sole (<i>Parophrys vetulus</i>). Isolates were evaluated using antimicrobial susceptibility typing, whole-genome sequencing, <i>fumC</i>, and multilocus sequence typing. Resistance and virulence genes were identified from sequence data. Of the 305 isolates from Salish Sea samples, 20 (6.6%) of the <i>E. coli</i> were intermediate, and 31 (10.2%) were resistant to ≥1 class of antibiotics, with 26.9% of nonsusceptible (resistant and intermediate resistant) <i>E. coli</i> isolates from marine mammals and 70% from river otters. The proportion of nonsusceptible isolates from animals was significantly higher than samples taken from marine water (<i>p</i> < 0.0001). A total of 196 unique STs was identified including 37 extraintestinal pathogenic <i>E. coli</i> (ExPEC)-associated STs [ST10, ST38, ST58, ST69, ST73, ST117, ST131, and ST405]. The study suggests that animals may be potential sentinels for antibiotic-resistant and ExPEC <i>E. coli</i> in the Salish Sea ecosystem.