In recent years, the landscape of highly pathogenic avian influenza (HPAI) virus infections has shifted, as 37 evidenced by an increase in infections among mammals. This includes the recent circulation of H5N1 in 38 dairy cattle herds in the USA and a rise in associated human cases. In this study, we investigated 39 differences in tissue tropism of two HPAI H5N1 strains, the isolate A/Vietnam/1203/2004 (VN1203) 40 isolated from a fatal human case in 2004 and the bovine isolate A/Bovine/Ohio/B24osu-342/2024 41 (Bov342) isolated in 2024, in C57BL/6J mice. Infection with either HPAI H5N1 isolate was uniformly 42 lethal in mice. However, tissue tropism differed significantly: while VN1203 replication was largely 43 restricted to the respiratory tract, Bov342 successfully replicated in the respiratory tract as well as various 44 regions of the brain. Bov342-challenged animals exhibited clinical signs consistent with central nervous 45 system (CNS) infection, and infectious virus was detected in brain tissue. Correspondingly, cytokine 46 profiles in the brain differed significantly between the isolates. Notably, in addition to abundant evidence 47 of CNS infection in Bov342-challenged mice via immunohistochemistry, sporadic intranuclear and 48 intracytoplasmic immunoreactivity was observed in other tissues in the head, including the choroid 49 plexus, retina, and inner ear. This study demonstrates that while both HPAI H5N1 isolates are uniformly 50 lethal in C57BL/6J mice upon aerosol exposure, significant differences exist in tissue tropism, with 51 Bov342 resulting in respiratory disease as well as increased neurotropism and inflammation in the brain 52 and nasal turbinates compared to VN1203, which predominantly induces respiratory disease.