The effects of weather/season (winter and summer), milk production (high and low), and milking time (night and day) on melatonin levels were evaluated. The melatonin in the milk from 30 individual cows, the bulk tank milk (BTM) from 16 farms, and ultra-high temperature (UHT) processed milk of 12 brands over two seasons were also assessed. The melatonin average concentration was 6.98 pg/mL in the cows' milk, 4.71 pg/mL in the BTM, and 5.62 pg/mL in the UHT milk. In the night milk, the melatonin concentration averaged 14.87 pg/mL, whereas it was 6.98 pg/mL in the total daily milk. In winter, the milk melatonin concentration was, on average, 74.7% higher than in the summer (13.89 vs. 7.95 pg/mL). The night milk from the low production group in winter also had a higher concentration of melatonin (41.94 pg/mL). The variables related to illuminance, such as the time of milking and season of the year, had considerable effects on the melatonin concentration in milk. Collecting and marketing melatonin-rich milk could benefit populations and agribusiness. ARTICLE HISTORY