Background: Nicotinamide riboside (NR) is a recently discovered NAD⁺ precursor vitamin with a unique biosynthetic pathway. Although the presence of NR in cow milk has been known for more than a decade, the concentration of NR with respect to the other NAD⁺+ precursors was unknown. Objective: We aimed to determine NAD⁺+ precursor vitamin concentration in raw samples of milk from individual cows and from commercially available cow milk. Methods: LC tandem mass spectrometry and isotope dilution technologies were used to quantify NAD⁺+ precursor vitamin concentration and to measure NR stability in raw and commercial milk. Nuclear magnetic resonance (NMR) spectroscopy was used to test for NR binding to substances in milk. Results: Cowmilk typically contained ~12 μmol NAD⁺+ precursor vitamins/L, ofwhich 60%was present as nicotinamide and 40% was present as NR. Nicotinic acid and other NAD⁺+ metabolites were below the limits of detection. Milk from samples testing positive for Staphylococcus aureus contained lower concentrations of NR (Spearman ρ = -0.58, P = 0.014), and NR was degraded by S. aureus. Conventional milk contained more NR than milk sold as organic. Nonetheless, NR was stable in organic milk and exhibited an NMR spectrum consistent with association with a protein fraction in skim milk. Conclusions: NR is a major NAD⁺+ precursor vitamin in cow milk. Control of S. aureus may be important to preserve the NAD⁺+ precursor vitamin concentration of milk.