AbstractIn vitro digestibility of protein was measured with pepsin/pancreatin in 321 spring barley lines grown in the field. The variation in digestibility was far less than the variation in the protein content. A small environmental influence on the digestibility was found. Two entries had slightly impaired digestibilities; these findings were partially verified in a repeated field trial, but were not confirmed in vivo. In vitro digestibilities of barleys grown in pots at various N‐levels were positively correlated with protein or hordein content. In vitro digestibility was negatively correlated with the fibre content in decortification fractions of Bomi and the high‐lysine mutant Risø 1508. The digestibility was positively correlated with the hordein concentration for the Bomi fractions, but not for the 1508 fractions.