The aim of the study was to determine the effect of substituting maize grain with barley grain in the diet of lactating Jersey cows grazing kikuyu-ryegrass pasture. Sixty Jersey cows were blocked in terms of number of days in milk, lactation number, milk yield and live weight and randomly assigned to one of five treatments (n = 12). The five treatments consisted of concentrate mixtures (12 MJ ME per kg/dry matter (DM), 130 g crude protein (CP)/kg DM) containing maize grain and barley grain at ratios of 100:0, 75:25, 50:50, 25:75 and 0:100. The cows received 6 kg (as fed basis) of the concentrate mixture a day, 3 kg during morning milking and 3 kg during afternoon milking. The investigation was conducted over a period of 42 days (14-day adaptation and 28-day measurement period). The cows strip-grazed an irrigated kikuyu-ryegrass pasture (157 ± 17.7 g DM/kg; 202 ± 42.6 g CP/kg DM; 447 ± 40.3 g neutral detergent fibre/kg DM). Milk yields were recorded daily. Milk samples were collected weekly and analyzed for milk fat and protein content. Body condition scores and live weights were recorded at the start and end of the experimental period. Milk production four weeks prior to the experiment was used as co-variate. The cows receiving the concentrate mixture containing an equal proportion of maize and barley grain (50:50) produced significantly more milk (17.0 kg/day) than those receiving the concentrate mixtures containing pure maize grain (15.7 kg/day) or pure barley grain (14.9 kg/day). Milk fat percentage, milk fat yield, protein percentage, live weight change and body condition score were not affected when barley grain substituted maize grain in the concentrate mixture. These results suggested that barley grain could be a successful substitute for maize grain in concentrates fed to Jersey cows grazing cultivated pasture in the Southern Cape region of South Africa.