Synchronization of the degradability of non-structural carbohydrate and rumen degradable protein has been identified as an effective method of increasing intestinal AA flow through increased microbial protein synthesis and more efficient ruminal fermentation, thereby increasing performance of dairy cows. Therefore, the objective was to determine the performance of lactating cows fed either corn and barley starches at varying proportions in diets containing canola meal as the major source of supplemental protein. Twelve multiparous and 4 primiparous Holstein cows (94 ± 25 DIM) were used in a 4 × 4 Latin square design with 28-d periods. The ratio of starch from ground corn and rolled barley within each treatment was 100:0, 67:33, 33:67, and 0:100. Diets contained 36% corn silage, 20% alfalfa haylage, and 44% concentrate (DM basis). Varying proportions of corn and barley had no effect (P > 0.10) on dry matter intake (26.5 kg/d) or milk production (41.2 kg/d). Milk fat percentage (3.52%) and yield (1.42 kg/d) and milk protein percentage (2.95%) and yield (1.21 kg/d) were not affected by starch. Lactose percentage (4.86, 4.83, 4.90, and 4.88%, for 100:0, 67:33, 33:67, and 0:100, respectively) and MUN (14.8, 14.5, 15.4, and 15.1 mg/dL) responded cubically (P < 0.05) to the changes in dietary starch proportions. Treatments did not affect energy-corrected milk (40.6 kg/d) nor feed efficiency (1.53). Increasing the proportion of barley to corn had no effect on the molar proportion of ruminal acetate and butyrate, however, propionate increased quadratically as barley increased in the diets (P < 0.01). Ruminal ammonia concentration averaged 11.5 mg/dl and was not affected by starch source. Apparent total-tract digestibilities of DM, OM, and NDF decreased linearly (P < 0.05) and, CP and ADF tended to decrease linearly (P < 0.10) when the proportion of barley starch increased in the diet. Total-tract digestibility of starch was not affected by starch source and averaged 95.5%. Overall, lactation performance was not affected by feeding varying proportions of corn and barley when the diets were formulated with canola meal as the primary protein supplement.