Transmission routes of highly pathogenic H5N1 between cows or to humans remain unclear due to limited data from affected dairy farms. We performed air, farm wastewater, and milk sampling on 14 H5N1-positive dairy farms across two different California regions. Infectious virus was detected in the air in milking parlors and in wastewater streams, while viral RNA was found in exhaled breath of cows. Sequence analysis of infectious H5N1 virus from air and wastewater samples on one farm revealed viral variants relevant for potential human susceptibility. Longitudinal analysis of milk from the individual quarters of cows revealed a high prevalence of subclinical H5N1-positive cows. Additionally, a heterogeneous distribution of infected quarters that maintained a consistent pattern over time was observed, inconsistent with shared milking equipment serving as the sole transmission mode. The presence of subclinically infected cows was further supported by detection of antibodies in the milk of animals that exhibited no clinical signs during the H5N1 outbreak on one farm. Our data highlight additional sources and potential modes of H5N1 transmission on dairy farms.