The aim of the study was to assess acid whey separated during traditional manufacture of tvarog – Polish acid curd cheese. Two variants of starter cultures were used in the study. The tvarog was made from bulk milk obtained from two breeds of cows (Polish Holstein-Friesian and Simmental) during two production seasons (spring/summer and autumn/ winter). Forty whey samples were analysed. The following parameters were determined in each sample: content of crude protein, fat, lactose, and dry matter, pH value, content of fat-soluble vitamins. The volume of whey obtained was determined as well. The breed of cow, season, and starter cultures were shown to influence the amount of whey separated as well as its chemical composition. The quantity of whey separated was significantly lower, which is technologically more favourable, in the production of tvarog from the milk of Simmental cows and in the spring/ summer, when the cows grazed in the pasture. The starter culture also influenced the separation of whey, with less whey obtained when starter variant 2 was used. Whey obtained in the spring and summer, irrespective of the cow breed, had lower protein and fat content, with higher content of vitamins. The starter cultures did not affect the composition of the whey.