The dairy industry plays an important role in the economy and food security of India. A study of the dairy value chains was conducted in Punjab, India, to identify production constraints and biosecurity risks. Focus group discussions and key informant interviews were conducted during 2018–2019 with a total of 119 participants comprising veterinarians (41), paraveterinarians (15), veterinary academics (12), dairy farmers (46) and key informants (5). Input and output value chains were created, and potential risk nodes were identified that could facilitate the transmission of pathogens between animals, farms and villages. The majority of the participants were male (93%), middle-aged (68%) or worked in rural areas (75%). Most of the farmers self-cultivated their green fodder (82%), used the wheat straw from their own fields (60%) but purchased commercial feed (63%). Artificial insemination was used by 85% of farmers for cattle, but only 68% for buffaloes. Most of the farmers (76%) reported getting their animals vaccinated against foot-and-mouth disease and hemorrhagic septicemia. Animals were sold and purchased without any health certification and testing in most cases. Adoption of biosecurity measures by farmers and the use of personal protective equipment by veterinary personnel were very low. We recommend conducting epidemiological studies to further characterize the identified risk nodes, training of veterinary practitioners and farmers to ensure adequate biosecurity practices and the appropriate use of personal protective equipment.