The objective of this experiment was to assess if feeding glyphosate-tolerant alfalfa affects feed intake, milk composition, or milk production of dairy cows. One alfalfa (Medicago sativa), variety expressing the CP4 EPSPS protein and grown in southeastern Washington State was harvested at the late vegetative stage as hay. Three commercial conventional varieties of alfalfa hay of similar nutrient composition and harvested in the same geographic region were fed to cows as controls. The commercial hays were selected to be similar in crude protein [18% of dry matter (DM)] and neutral detergent fiber (40% of DM) to the glyphosate-tolerant hay. Sixteen multiparous Holstein cows were fed diets containing alfalfa hay (39.7% of diet DM) from either the glyphosate-tolerant alfalfa, or 1 of the 3 conventional varieties. Diets contained at least 15.7% crude protein and 29% neutral detergent fiber. Experimental design was a replicated 4 x 4 Latin square. Periods were 28 d and feed intake, milk yield, and milk composition were summarized over the last 14 d of each period. Daily milk yield (38.0 kg) and 4% fat-corrected milk (34.7 kg) were not affected by treatment. Milk fat (3.44%) and milk true protein (2.98%) were also not affected by source of hay. Milk lactose (4.72%) and soldis-not-fat (8.5%) did not differ due to treatment. Dry matter intake was similar across treatments (24.4 kg/d). These results are consistent with data from feeding trials with other glyphosate-tolerant crops and previously reported compositional comparisons of glyphosate-tolerant alfalfa with controls. Milk production, milk composition, feed intake, and feed efficiency were not affected by feeding diets that contained nearly 40% glyphosate-tolerant alfalfa hay to lactating dairy cows.