The objective of the present study was to identify the factors associated with both the protein composition and free amino acid (FAA) composition of bovine milk predicted using mid-infrared spectroscopy. Milk samples were available from 7 research herds and 69 commercial herds. The spectral data from the research herds comprised 94,286 separate morning and evening milk samples; the spectral data from the commercial herds comprised 40,260 milk samples representing a composite sample of both the morning and evening milkings. Mid-infrared spectroscopy prediction models developed in a previous study were applied to all spectra. Factors associated with the predicted protein and FAA composition were quantified using linear mixed models. Factors considered in the model included the fixed effects of calendar month of the test, milking time (i.e., morning, evening, or both combined), parity (1, 2, 3, 4, 5, and ≥6), stage of lactation, the interaction between parity and stage of lactation, breed proportion of the cow (Friesian, Jersey, Norwegian Red, Montbéliarde, and other), and both the general heterosis and recombination coefficients of the cow. Contemporary group as well as both within- and across-lactation permanent environmental effects were included in all models as random effects. Total proteins (i.e., total casein, CN; total whey; and total β-lactoglobulin) and protein fractions (with the exception of α-lactalbumin) decreased postcalving until 36 to 65 days in milk and increased thereafter. After adjusting the statistical model for differences in crude protein content and milk yield separately, irrespective of stage of lactation, younger animals produced more total proteins (i.e., total CN, total whey, and total β-lactoglobulin) as well as more total FAA, Glu, and Asp than their older contemporaries. The concentration of all protein fractions (except β-CN) in milk was greatest in the evening milk, even after adjusting for differences in the crude protein content of the milk. Relative to a purebred Holstein cow, Jersey cows, on average, produced a greater concentration of all CN fractions but less total FAA, Glu, Gly, Asp, and Val in milk. Relative to their respective purebred parental average, first-cross cows produced more total CN and more β-CN. Results from the present study indicate that many cow-level factors, as well as other factors, are associated with protein composition and FAA composition of bovine milk.