Conflicting reports of the quantities of biologically active compounds present in milk from organic grass-fed and conventional herds show that more research is required, especially as these compounds are linked to human health benefits and can improve the health value consumers place on dairy products. In collaboration with the Rodale Institute, Kutztown, Pennsylvania, a 3-year study evaluated the composition of milk obtained from 2 farms adjacent to the Rodale experimental farm: one herd transitioned to certified organic in the first year; cows averaged over 50% dry matter intake from pasture during the grazing season. The other herd was on a confined conventional farm with no access to pasture. This study provided a unique opportunity to compare milk from farms of similar soil types, climate, and weather. Over a 150-week period (3 grazing seasons), weekly milk samples were collected and assayed for composition and physical traits, and profiled for fatty acids (FAs) and proteins-peptides. The overall protein, lactose, ash, minerals, total solids contents, pH, water activity, titratable acidity, and protein profiles of the milk samples from both farms were similar (P < 0.05). Fat content was more variable in the milk from the organic herd, especially after the seasonal grazing was implemented, with higher fat levels (P < 0.05) obtained during the winter months. The FA profiles were the major difference between the milk from the 2 herds. Compared with the confined herd, the grazing herd milk had considerable variation in the FA profiles, which generally stabilized after 4 mo, and had higher levels (P < 0.05) of α-linolenic acid (omega-3 FA) after the second month. Once certified, the organic herd produced milk that contained 36% more omega-3 FA and had lower linoleic acid (omega-6):omega-3 FA values throughout the year, and 25–30% more conjugated linoleic (CLA) during the grazing seasons than the confined herd. Because higher CLA and omega-3 FA levels and a low omega-6:omega-3 value have been linked to being beneficial to human health, the importance of pasture grazing must be considered in supplying milk and dairy products that address consumers’ demand for foods that support human health and wellness.