Powassan virus lineage 2 (deer tick virus) is an emergent threat to American public health, causing severe neurologic disease. Its life cycle in nature remains poorly understood. We use a host-specific retrotransposon-targeted real time PCR assay to test the hypothesis that white-footed mice, considered the main eastern U.S. reservoir of the coinfecting agent of Lyme disease, is the reservoir for deer tick virus. Of 20 virus-infected host-seeking nymphal black-legged ticks 65% fed on shrews and none on mice. The proportion of ticks feeding on shrews at a site is positively associated with prevalence of viral infection, but not the Lyme disease agent. Viral RNA is detected in the brain of one shrew. We conclude that shrews are a likely reservoir host for deer tick virus and that host bloodmeal analysis can provide direct evidence to incriminate reservoir hosts, thereby promoting our understanding of the ecology of tick-borne infections.