A highly pathogenic avian influenza (HPAI) H5N1 virus belonging to clade 2.3.4.4b has been detected in dairy cattle in the United States, marking a significant spillover event. The virus caused clinical signs in affected cows, including reduced feed intake, respiratory distress, and decreased milk production. Viral RNA and infectious virus were consistently found in milk, and the virus showed a distinct tropism for the epithelial cells lining the mammary gland. Whole-genome sequencing of the virus from dairy cows, birds, cats, and a raccoon indicated multidirectional interspecies transmission. Epidemiological and genomic data revealed efficient cow-to-cow transmission after apparently healthy cows were transported to a different state. The virus was also detected in various mammalian species, including cats, raccoons, and seals, and in wild birds. The virus was found to spread between farms, with evidence of transmission between farms and between cattle and other species. The HPAI H5N1 virus was detected in milk and tissues of affected cattle, with high viral RNA loads in milk. The virus was also detected in the central nervous system of cats, indicating a potential for cross-species transmission. The virus was found to have a distinct tropism for the mammary gland of cattle and the central nervous system of cats. The virus was also detected in other tissues, including the lung, lymph nodes, and spleen. The virus was found to have undergone reassortment, incorporating gene segments from different avian lineages. The virus was detected in multiple farms across the United States, with evidence of transmission between farms and between cattle and other species. The results highlight the ability of the virus to cross species barriers and the potential for sustained transmission in mammals. The study underscores the need for enhanced biosecurity and surveillance in affected and non-affected farms.A highly pathogenic avian influenza (HPAI) H5N1 virus belonging to clade 2.3.4.4b has been detected in dairy cattle in the United States, marking a significant spillover event. The virus caused clinical signs in affected cows, including reduced feed intake, respiratory distress, and decreased milk production. Viral RNA and infectious virus were consistently found in milk, and the virus showed a distinct tropism for the epithelial cells lining the mammary gland. Whole-genome sequencing of the virus from dairy cows, birds, cats, and a raccoon indicated multidirectional interspecies transmission. Epidemiological and genomic data revealed efficient cow-to-cow transmission after apparently healthy cows were transported to a different state. The virus was also detected in various mammalian species, including cats, raccoons, and seals, and in wild birds. The virus was found to spread between farms, with evidence of transmission between farms and between cattle and other species. The HPAI H5N1 virus was detected in milk and tissues of affected cattle, with high viral RNA loads in milk. The virus was also detected in the central nervous system of cats, indicating a potential for cross-species transmission. The virus was found to have a distinct tropism for the mammary gland of cattle and the central nervous system of cats. The virus was also detected in other tissues, including the lung, lymph nodes, and spleen. The virus was found to have undergone reassortment, incorporating gene segments from different avian lineages. The virus was detected in multiple farms across the United States, with evidence of transmission between farms and between cattle and other species. The results highlight the ability of the virus to cross species barriers and the potential for sustained transmission in mammals. The study underscores the need for enhanced biosecurity and surveillance in affected and non-affected farms.