Oat (Avena sativa L.) β-glucan lowers serum cholesterol in humans. Development of oat cultivars with greater groat (caryopsis) β-glucan content would increase the nutritional and economic value of the crop. The objectives of this experiment were to evaluate the response to phenotypic selection among individual S0 plants for greater groat β-glucan content in two genetically broad-based populations; to compare selected experimental lines to standard check cultivars; and to estimate genetic variances and heritabilities and to test for nonadditive genetic variance for β-glucan content. We measured groat β-glucan contents of check cultivars and parental lines and random S0:1 lines from initial and selected generations of each population grown in field experiments in 1996 and 1997 at two Iowa locations. Mean β-glucan content increased from 53.9 to 59.9 g kg−1 in one population, and from 63.5 to 66.0 g kg−1 in the other, following selection. Genetic variance of β-glucan content decreased by 9 to 22% following selection, but heritability for β-glucan content did not change significantly. Heritability estimates ranged from 0.80 to 0.85 on a line mean basis. Additive variance was the only substantial component of genetic variance. Some experimental lines had significantly greater β-glucan content than the best check cultivars and lines. Phenotypic selection for greater groat β-glucan content will be effective for developing cultivars with elevated β-glucan contents.