Optimizing cellulosic ethanol yield depends strongly on understanding the biological variation of feedstocks. Our objective was to study variation in capacity for producing fermentable sugars from straw of winter wheat cultivars with a high-throughput pretreatment and hydrolysis well-plate technique. This technique enabled us to estimate cultivar-related and environmental correlations between sugar yield, chemical composition, agronomic qualities, and distribution of botanical plant parts of wheat straw cultivars. Straws from 20 cultivars were collected in duplicates on two sites in Denmark. Following hydrothermal pretreatment (180 °C for 17.6 min) and co-hydrolysis, sugar release and sugar conversion were measured. Up to 26% difference in sugar release between cultivars was observed. Sugar release showed negative cultivar correlation with lignin and ash content, whereas sugar release showed positive cultivar correlation with content of carbohydrates and plant height. Accessibility to cellulose can impede the sugar conversion rate, and convertibility of each botanical fraction might be more important to overall sugar conversion than the relative proportions of botanical fractions. Our results suggest that selection of cultivars for improved biofuel feedstock of wheat straw is possible, because heritability of sugar release is 57% and there are few adverse correlations to other agronomic traits.