Aleutian mink disease virus (AMDV) occurs in the American mink (Neovison vison) in wild populations and on mink farms and can cause illness and death. The North American river otter (Lontra canadensis) may be exposed to AMDV because of shared space and habitat with mink. Using serology and real-time PCR, we tested river otters across Ontario for AMDV infection. We found no evidence of infection in otters, a surprising finding given the sympatric distribution, niche overlap, and close phylogenetic relationship of the river otter and the American mink. Our results are consistent with the hypothesis that the major point of spillover of AMDV between mink farms and wildlife is manure and composting carcasses on mink farms. Mink farms in Ontario are generally in agricultural landscapes; it is unlikely that river otter use these habitats and thus are likely not exposed to AMDV. We found no evidence that AMD is an important disease for the river otters in Ontario.