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Background: Throughout the Americas, Lyssavirus rabies (RV) perpetuates as multiple variants among bat and mesocarnivore species. Interspecific RV spillover occurs on occasion, but clusters and viral host shifts are rare. The spillover and host shift of a big brown bat (Eptesicus fuscus) RV variant Ef-W1 into mesocarnivores was reported previously on several occasions during 2001–2009 in Flagstaff, Arizona, USA, and controlled through rabies vaccination of target wildlife. During autumn 2021, a new cluster of Ef-W1 RV cases infecting striped skunks (Mephitis mephitis) was detected from United States Department of Agriculture enhanced rabies surveillance in Flagstaff. The number of Ef-W1 RV spillover cases within a short timeframe suggested the potential for transmission between skunks and an emerging host shift. Materials and Methods: Whole and partial RV genomic sequencing was performed to evaluate the phylogenetic relationships of the 2021–2023 Ef-W1 cases infecting striped skunks with earlier outbreaks. Additionally, real-time reverse-transcriptase PCR (rtRT-PCR) was used to opportunistically compare viral RNA loads in brain and salivary gland tissues of naturally infected skunks. Results: Genomic RV sequencing revealed that the origin of the 2021–2023 epizootic of Ef-W1 RV was distinct from the multiple outbreaks detected from 2001–2009. Naturally infected skunks with the Ef-W1 RV showed greater viral RNA loads in the brain, but equivalent viral RNA loads in the mandibular salivary glands, compared to an opportunistic sample of skunks naturally infected with a South-Central skunk RV from northern Colorado, USA. Conclusion: Considering a high risk for onward transmission and spread of the Ef-W1 RV in Flagstaff, public outreach, enhanced rabies surveillance, and control efforts, focused on education, sample characterization, and vaccination, have been ongoing since 2021 to mitigate and prevent the spread and establishment of Ef-W1 RV in mesocarnivores. [ABSTRACT FROM AUTHOR]