Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are spread by mosquitoes and cause human disease and mortality in tropical areas. In contrast, Powassan virus (POWV), which causes severe neurologic illness, is a flavivirus transmitted by ticks in temperate regions of the Northern hemisphere. We find serologic neutralizing activity against POWV in individuals living in Mexico and Brazil. Monoclonal antibodies P002 and P003, which were derived from a resident of Mexico (where POWV is not reported), neutralize POWV lineage I by recognizing an epitope on the virus envelope domain III (EDIII) that is shared with a broad range of tick- and mosquito-borne flaviviruses. Our findings raise the possibility that POWV, or a flavivirus closely related to it, infects humans in the tropics. Display omitted •Sera from Mexico and Brazil neutralize POWV and TBEV, which are not known to circulate there•Monoclonal antibody P014 binds to POWV but not to endemic flaviviruses•Monoclonal antibodies P002 and P003 neutralize POWV lineage I•The P003 epitope is shared across 13 flaviviruses from tick and mosquito serocomplexes Cervantes Rincón et al. describe antibody neutralizing activity against Powassan virus from regions of the Americas where this flavivirus transmitted by ticks is not known to circulate. Two monoclonal antibodies to the EDIII (P002 and P003) are broadly cross-reactive against tick- and mosquito-borne flaviviruses.