Simple SummaryAnimal breeding is one of the most profitable human activities. The ultimate goal is to join genetically high performance and good health. In the last decades, the study of genetic control of important traits on the molecular level has been launched. In the study of milk performance, major genes, namely those for milk proteins, are the focus of much research. Here, we studied the polymorphisms ofCSN1S1andLALBAgenes and their associations with milk performance and quality. Also, other non-genetic effects were evaluated. The polymorphisms influenced milk, fat, and protein yield percentages in the second lactation but their effects in the first lactation were not significant. The technological properties of milk were not influenced. The effect of the farm was significant in most cases. Breed influenced milk performance but did not influence technological properties. Changes in lactose content reflect the health of the udder.AbstractWe evaluated the influence ofCSN1S1andLALBApolymorphisms on cow’s milk yield and quality. The analysis was done on Czech Simmental and Holstein cows. Non-genetic factors were included as well.CSN1S1did not influence the milk performance in the first lactation. In the second lactation, cows with theBBgenotype had significantly higher milk, protein, and fat yields thanBC. The differences betweenLALBAgenotypes were non-significant in the first lactation, while in the second lactation, the fat percentage was significantly higher inBBthan inAB. 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 ofCSN1S1andLALBAgenotypes 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.