BACKGROUND: During 2017, a multi-state outbreak investigation occurred following the confirmation of Seoul virus (SEOV) infections in people and pet rats. A total of 147 humans and 897 rats were tested. METHODS: In addition to IgG and IgM serology and traditional RT-PCR, novel quantitative RT-PCR primers/probe were developed, and whole genome sequencing was performed. RESULTS: Seventeen people had SEOV IgM, indicating recent infection; seven reported symptoms and three were hospitalized. All patients recovered. Thirty-one facilities in 11 US states had SEOV infection, and among those with >/=10 rats tested, rat IgG prevalence ranged 2-70% and SEOV RT-PCR positivity ranged 0-70%. Human lab-confirmed cases were significantly associated with rat IgG positivity and RT-PCR positivity (p=0.03 and p=0.006, respectively). Genomic sequencing identified >99.5% homology between SEOV sequences in this outbreak, and these were >99% identical to SEOV associated with previous pet rat infections in England, the Netherlands, and France. Frequent trade of rats between home-based ratteries contributed to transmission of SEOV between facilities. CONCLUSIONS: Pet rat owners, breeders, and the healthcare and public health community should be aware and take steps to prevent SEOV transmission in pet rats and to humans. Biosecurity measures and diagnostic testing can prevent further infections.