The aim of this study was to analyze milk protein composition in purebred and crossbred dairy cattle and estimate the effects of individual sources of variation on the investigated traits. Milk samples were collected from 505 cows from three commercial farms located in Northern Italy, some of which had originated from crossbreeding programs, although most were purebred Holsteins (HO). The basic crossbreeding scheme was a three-breed rotational system using Swedish Red (SR) semen on HO cows (SR×HO), Montbeliarde (MO) semen on SR×HO cows (MO×(SR×HO)) and HO semen again on MO×(SR×HO) cows. A smaller number of purebred HO from each of the herds were mated inverting the breed order (MO×HO and SR×(MO×HO)) or using Brown Swiss (BS) bulls (BS×HO) then MO bulls (MO×(BS×HO)). Milk samples were analyzed by reverse-phase HPLC to obtain protein fraction amounts (g/l) and proportions (% of total true protein). Traits were analyzed using a linear model, which included the fixed effects of herd-test-day (HTD), parity, days in milk and breed combination. Results showed that milk protein fractions were influenced by HTD, stage of lactation, parity and breed combination. The increase in protein concentration during lactation was due in particular to β-casein (β-CN), αS1-CN and β-lactoglobulin (β-LG). The higher protein content of primiparous milk was mainly due to higher concentrations of all casein fractions. The milk from crossbred cows had higher contents and proportions of κ-CN and α-lactalbumin (α-LA), lower proportions of β-LG and greater proportion of caseins/smaller in whey proteins on milk true protein than purebred HO. The three-way crossbreds differed from two-way crossbreds only in having greater proportions of α-LA in their milk. Of the three-way crossbreds, the SR sired cows yielded milk with a smaller content and proportion of β-LG than the MO sired cows, and, consequently, a higher proportion of caseins than whey proteins. Results from this study support the feasibility of using crossbreeding programs to alter milk protein profiles with the aim of improving milk quality and cheese-making properties.