In recent years, beef industry has improved production processes to ensure quality and certification for overseas meat markets. However, there is limited scientific information about the nutritional quality of beef from Chile. The aim of the present study was to evaluate the nutritional quality of beef produced in Chile from different production systems classified according to the type of finishing diet. Two-hundred and five animals from 13 livestock farms were used, 80 steers were finished on grazing pasture, 79 on pasture supplemented with grain or concentrate and 46 steers were finished on a typical feedlot system in the central zone of Chile. Longissimus thoracis intramuscular fat, fatty acid composition and cholesterol content were determined. Beef from pasture-fed animals showed higher content of vaccenic acid (C18:1 trans-11) rumenic acid (CLA cis-9 trans-11) and n-3 fatty acids, and lower n-6:n-3 ratio than beef from feedlot animals. However, Chilean beef from the three production systems showed similar intramuscular fat percentages and a n-6:n-3 ratio lower than 4.0. Results indicate that the Longissimus muscle from Chilean beef could be recommended as a source of red meat for a healthy diet.