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The impact of genotype, environment, and rotation on the variability of important bioactive compounds (protein and β‐glucan) was evaluated in the grains of seven naked and two husked oat (Avena sativa L.) genotypes, grown in a set of trials in 2001–2002 and 2002–2003 in two Italian locations in winter seeding. Each trial was composed of two experiments, corresponding to different rotation systems, i.e., cereal or legume as the preceding crop. Genetic, agronomic, and environmental effects for all the parameters investigated and significant interactions between variability factors were found. Plots grown after cereal appeared to accumulate more protein (+4%) and β‐glucan (+9%) than those grown after legume. Significant interactions were found between rotation and genotypes. Total dietary fiber (TDF) content was also determined in a group of samples and found to be mostly determined by genotype and its interaction with other factors. Among the naked genotypes, ‘Bikini’ and Hja72095N showed the highest protein content; ‘Abel’, ‘Nave’, and Bikini were characterized by high β‐glucan content, and ‘Konradin’ had the largest amount of TDF in all environments. The present paper furnishes useful indicators to identify the most appropriate genotypes and the correct cultural practices to improve the nutritional value of naked oats, making them a suitable source of raw material for innovative food production.

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