We evaluated the influence of CSN1S1 and LALBA polymorphisms on cow’s milk yield and quality. The analysis was done on Czech Simmental and Holstein cows. Non-genetic factors were included as well. CSN1S1 did not influence the milk performance in the first lactation. In the second lactation, cows with the BB genotype had significantly higher milk, protein, and fat yields than BC. The differences between LALBA genotypes were non-significant in the first lactation, while in the second lactation, the fat percentage was significantly higher in BB than in AB. The farm significantly influenced milk, protein, and fat yields in both the first and second lactations and fat percentage in the first lactation. The effect of CSN1S1 and LALBA genotypes on the milk technological quality was non-significant. Breed did not influence any of the evaluated technological traits and SCS. The ethanol test was not influenced by farm, season, lactation phase, protein percentage, breed, or non-fat solids percentage. Farm, season, and protein percentage significantly influenced milk fermentation ability, renneting, and SCS. The lactose content is a good indicator of udder health.