Our objective was to determine the relationship between bulk tank fat and protein concentration and the concentration of denovo (C4 to C14), mixed origin (C16 and C16:1), and preformed fatty acids (>C18) expressed as g per 100 g milk and milk fat unsaturation expressed as double bonds per fatty acid. Bulk tank milks from 46 Holstein farms were collected and analyzed for fat, true protein, and milk fatty acid composition determined using mid-IR milk analysis over a period of 16 mo with a Delta FTA mid-IR milk analyzer. Milk sample collection was done by the licensed milk truck driver as part of the official sampling for payment testing at the St Albans Cooperative (St Albans, Vermont) and the samples in the study were the same samples used to determine fat and protein for payment. The number of milk samples analyzed per farm per month ranged from a minimum of 3 to as many as 20 within a month. Farms producing more milk per day were sampled more frequently. Mean monthly average bulk tank fat concentration (Y) increased (P < 0.05) with increased de novo fatty acid concentration (X) (Y = 2.297X + 1.844; r² = 0.80) and increased mixed origin fatty acids (Y = 1.5396X + 1.5896; r² = 0.88), while the bulk tank milk fat concentration was not related to concentration of preformed fatty acids (r² = 0.07). Milk fat (Y = −8.583X + 6.4213; r² = 0.69) and true protein (Y = −2.5588X + 3.8812; r² = 0.34) concentration both decreased (P < 0.05) with increasing milk fatty unsaturation expressed as double bonds per fatty. Bulk tank milk true protein concentration increased with increasing de novo fatty acid concentration (Y = 0.8005X + 2.4179; r² = 0.53). For a Holstein farm to achieve a >3.80% bulk tank fat test, the denovo fatty acid concentration needs to be >0.85 g/100g milk and the double bonds per fatty acid should be < 0.305 double bonds per fatty acid. Higher denovo and mixed origin fatty acid concentration was related to higher milk fat and true protein concentration while preformed fatty acids was not.