Marine vesiviruses were isolated in cell culture from oral and rectal swabs and vesicular fluid from Alaskan Steller sea lions (SSL; Eumetopias jubatus). Further characterization by RT-PCR, complete genomic sequencing, and phylogenetic analyses indicated that these viruses are most closely related to the marine vesiviruses, but are distinct viruses and represent two novel genotypes. The complete genome of these two SSL isolates was sequenced after cloning their viral cDNA. The genomes were found to be 8302 and 8305 nucleotides in length, organized in three open reading frames and contained 5' and 3' untranslated regions (UTR) of 19 and 180 nucleotides, respectively. The complete genomes of both SSL viruses were most closely related to each other and shared 83.0% nucleotide identity. Using the very limited number of complete genomic vesivirus sequences available in the NCBI database, these novel SSL vesiviruses seem most closely related to vesicular exanthema of swine virus-A48 and least related to rabbit vesivirus and walrus calicivirus. Specific antiserum against some evolutionary closer marine vesiviruses did not neutralize these isolates supporting the novel nature of these SSL viruses.