Transmission routes of highly pathogenic H5N1 between cows or to humans remain unclear due to limited data from affected dairy farms. We performed extensive air, farm wastewater, and milk sampling on 14 H5N1 positive dairy farms across two different California regions. Virus was detected in the air in milking parlors and from exhaled breath of cows. Infectious H5N1 virus was detected in the air and water streams; sequence analysis revealed viral variants on a farm in these locations. Longitudinal analysis of milk from the individual quarters of cows revealed a high prevalence of subclinical H5N1 positive cows and a heterogeneous distribution of infected quarters that maintained a consistent pattern over time. Our data highlight potential modes of H5N1 transmission on dairy farms.