The rennet coagulation of milk is a two-step process: the primary phase is the enzymatic hydrolysis of the κ-casein that is present on the surface of casein micelles, while the secondary phase involves the aggregation and gelation of destabilized micelles. Milk coagulation is a crucial step in cheesemaking but substantial variations in milk coagulation properties have been found among individual bovine milk. The aim of the present study was to provide further knowledge of the effects of κ-casein phenotypes on κ-casein fractions and milk coagulation properties from individual raw milk samples from one farm (same feeding). The analysis of κ-casein variants was directly performed on amino acids in the milk samples using a newly developed LC-MS method. The κ-casein variants had no direct effect on casein concentrations or on any main ingredient of milk but were important for κ-casein fractions and firmness during the coagulation step. κ-casein might play a fundamental role in the primary phase of enzymatic hydrolysis (rennet-induced coagulation), and it was suggested that this process is highly connected to the genetic κ-casein variants.