The study involved 68 farms located in 3 regions of Eastern Poland, i.e. in the lowland (terrains near the Bug, Biebrza, and Narew rivers), in the Beskid Niski Mountains, and in the Bieszczady Mountains. The farms studied were arranged in 2 groups: 1 group were those producing milk in an intensive system, and the second group: those using a traditional system. In 1589 milk samples, the following was determined: contents of fat, protein, casein, lactose, and dry matter; acidity (pH), heat stability; rennet coagulation time; contents of α-lactalbumin, β-lactoglobulin, serum albumin, lactoferrin, and lysozyme. In the selected 298 samples, a fatty acid profile was also determined. It was found that the milk production system (intensive vs. traditional) had a significant (p ≤ 0.01 and p ≤ 0.05) impact on the basic chemical composition, concentration level of whey proteins, fatty acid profile, acidity, rennet coagulation time, and heat stability. The effect of region where milk was produced was less marked; however, in many cases, it was statistically significant. The milk manufactured in the Beskid Niski M. region had the highest contents of basic components as well as of some fractions of whey proteins. This could be attributed to the dominant cow breed (Polish Red) in this region and to a higher diversity of botanical composition of meadow and pasture forages. Significant (p ≤ 0.001) interactions between the two analyzed factors (i.e. production system and production region) were found in nearly all evaluated basic milk components and whey proteins fractions.