Toxoplasmosis in marine mammals is epidemiologically and clinically important. Toxoplasma gondii antibodies (by modified agglutination test, cut-off ≥1:25) were detected in serum of 65 of 70 (92.9%) northern sea otters (Enhydra lutris kenyoni) from Washington State, USA. Brains and/or muscles of 44 sea otters were bioassayed in mice (INF-γ knock-out [KO], Swiss Webster outbred [SW]) and viable T. gondii was isolated from 22 of 44 (50%); T. gondii strains were lethal to KO mice but not SW mice. These T. gondii isolates were further propagated in cell culture. Multi-locus PCR-RFLP genotyping of cell culture-derived tachyzoites revealed four different genotypes among 22 isolates including ToxoDB PCR-RFLP genotype #5 (14 isolates), #1 (three isolates), #3 (four isolates), and #167 (one isolate). PCR-DNA sequencing based genotyping using polymorphic gene GRA6 revealed one of four different alleles. Among the 14 RFLP genotype #5 strains, 10 have GRA6 sequences that match with the Type A, one match with the Type X, two strains did not generate sequence data, and one strain had double peaks at known polymorphic sites indicating a mixed infection. The seven strains belong to genotypes #1 and #3, all have identical sequences to T. gondii Type II reference isolate ME49. Genotype #167 strain has identical sequence to Type I reference strain. In summary, we observed high seroprevalence, and high rate of isolation of T. gondii from northern sea otters and predominant genotype #5 that has been previously reported a dominant and widespread strain among terrestrial wildlife in North America. GRA6 sequence analysis of the genotype #5 isolates indicated the dominance of Type A lineage in sea otters in Washington State.