This study reports the spillover of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b virus into dairy cattle herds across several states in the United States. Affected cows exhibited clinical signs such as decreased feed intake, altered fecal consistency, respiratory distress, and reduced milk production with abnormal milk. Viral RNA and infectious virus were consistently detected in milk from affected cows. Immunohistochemistry and in situ hybridization revealed that the virus primarily infected the epithelial cells lining the alveoli of the mammary gland in cows. Whole genome sequencing from dairy cows, birds, domestic cats, and a raccoon from affected farms indicated multidirectional interspecies transmissions. Epidemiological and genomic data suggested efficient cow-to-cow transmission after apparently healthy cows from an affected farm were transported to a different state. These findings highlight the transmission of HPAI H5N1 clade 2.3.4.4b virus at a non-traditional interface, underscoring the virus's ability to cross species barriers. The study also discusses the potential routes of virus entry, infection dynamics, and pathogenesis in dairy cattle, emphasizing the need for enhanced biosecurity practices and surveillance efforts.This study reports the spillover of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b virus into dairy cattle herds across several states in the United States. Affected cows exhibited clinical signs such as decreased feed intake, altered fecal consistency, respiratory distress, and reduced milk production with abnormal milk. Viral RNA and infectious virus were consistently detected in milk from affected cows. Immunohistochemistry and in situ hybridization revealed that the virus primarily infected the epithelial cells lining the alveoli of the mammary gland in cows. Whole genome sequencing from dairy cows, birds, domestic cats, and a raccoon from affected farms indicated multidirectional interspecies transmissions. Epidemiological and genomic data suggested efficient cow-to-cow transmission after apparently healthy cows from an affected farm were transported to a different state. These findings highlight the transmission of HPAI H5N1 clade 2.3.4.4b virus at a non-traditional interface, underscoring the virus's ability to cross species barriers. The study also discusses the potential routes of virus entry, infection dynamics, and pathogenesis in dairy cattle, emphasizing the need for enhanced biosecurity practices and surveillance efforts.