Our objective was to determine the relationship between bulk tank or tanker milk fat and true protein concentration and the concentration of de novo (DN: C4 to C14), mixed origin (MO: C16 and C16:1), and preformed (PF: > C18) fatty acids (FA) expressed as g per 100 g milk. Bulk tank or tanker milk samples from 167 Holstein farms (50 to >10,000 cows/farm) were collected from a wide geographic area within the US and analyzed for fat, true protein, and milk FA composition determined using mid-infrared red (MIR) milk analysis with a Delta model FTA MIR milk analyzer. Bulk tank and tanker milk samples were preserved with Microtab II at 1 tablet per 90 mL milk, refrigerated, and shipped overnight on ice for testing. Dairy farms included both total mixed ration and grazing feeding systems. Herd average milk production per cow ranged from 22 to 47 kg per cow per d while concentrations of fat and true protein were from 3.0 to 4.3% and 2.75 to 3.35%, respectively. Mean milk fat concentration (Y) increased significantly (P < 0.05) with increased DN FA concentration (X) (Y = 2.241 X + 1.794; R² = 0.62) and increased MO FA concentration (X) (Y = 1.894 X + 1.184; R² = 0.77) when expressed as g FA/100 g milk. Bulk tank milk fat concentration increased with increasing PF FA concentration, but the relationship was not as strong (Y = 1.30 X + 1.89; R² = 0.37) as for DN and MO FA. Bulk tank milk true protein concentration increased with increasing DN FA concentration (Y = 0.714 X + 2.453; R² = 0.30). The relationships over a wide geographic area of the US and more diverse farm management practices were similar to those found in a previous study of Holstein herds in northeastern US. Using data from bulk tank or tanker milk, FA composition may be useful in understanding rumen fermentation driven changes in milk fat and protein concentration.