Our objective was to determine if there is a relationship between seasonal variation in bulk tank milk fat and protein concentration and the concentration of de novo (DN: C4 to C15), 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 46 Holstein farms was analyzed for fat, true protein, and milk fatty acid composition determined using mid-infrared red (MIR) milk analysis with a Delta model FTA MIR milk analyzer. Milk from each farm was analyzed between 6 and 25 times per month for the period of January 2014 through December 2017 as part of the routine milk payment testing at the St Albans Cooperative, St Albans, Vermont. A monthly average of the milk composition was calculated for 46 farms. The seasonal variation in fat and true protein content for the group of farms had the typical seasonal pattern with fat and true protein concentration being high in the winter months and low in the summer months. Seasonal variation of fat and true protein concentration (g/100 g milk) had a similar temporal pattern as DN + MO FA concentration (g/100 g milk), while this temporal pattern in relation to fat and true protein was not observed with variation in PF FA concentration . Mean milk fat concentration (Y) increased significantly (P < 0.05) with increasing DN + MO FA concentration expressed as g FA/100 g milk (X) (R-squared 0.60) and true protein concentration (X) (R-squared = 0.59). Bulk tank milk fat (R-squared 0.22) was increased significantly (P < 0.05), while no significant relationship (P > 0.05) between PF FA and true protein was observed. The typical decrease in milk fat and protein in the summer months may be related to factors that cause changes in rumen fermentation of carbohydrates and production of rumen volatile fatty acids (VFA) as reflected by lower de novo FA content of milk fat. Identification of seasonal factors influencing rumen VFA may lead to dairy cow feeding and management strategies that minimize seasonal variation in bulk milk composition for dairy product manufacture and allow more efficient production of dairy products.