Drinkwater, J. M., Tsao, R., Liu, R. and Wolyn, D. J. 2014. Genetic variation and heritability of rutin and glutathione concentrations in asparagus spears. Can. J. Plant Sci. 94: 1355-1362. Asparagus (Asparagus officinalis L.) spears contain numerous phytochemicals, including rutin (a flavonoid) and glutathione (a biothiol), that are beneficial to human health due to antioxidant or other activities. To assess the potential to increase phytochemical content through breeding, genetic variability was surveyed in diverse germplasm adapted to southern Ontario. Selected female and supermale genotypes were also hybridized in a partial diallel to estimate heritability and identify parents with good combining ability. Genetic variation for rutin (3.30 to 8.14 mg g^-1 dry weight) was observed, while glutathione concentrations (5.37 to 7.86 µmol g^-1 dry weight) did not differ significantly among tested genotypes. Combining ability effects were significant for rutin concentration and heritability was also moderate. From these results, breeding to improve the concentration of rutin, rather than that of glutathione, is most achievable in the germplasm tested.