Species hybridization is key for the improvement of shrub willow (Salix spp.) bioenergy crops, because hybrids often display heterosis for yield. Development of high-yielding genotypes requires numerous broad attempts at hybridization followed by field evaluation and selection for stable performance. Selection of improved shrub willow varieties for use as a bioenergy crop involves evaluation of full-sib progeny in family-based selection trials. Improving the accuracy of evaluation would greatly improve the efficiency of selection. Heterosis for biomass yield in intra- and interspecific F1 and F2 shrub willow (Salix spp.) was examined utilizing a suite of biomass, foliar, and physiological traits collected over the course of 12 weeks in the greenhouse and over two years in the field. Triploid families generated from diploid S. viminalis and tetraploid S. miyabeana displayed the greatest levels of heterosis for harvestable biomass and biomass-related growth traits in the greenhouse and in the field. While intraspecific S. purpurea diploids exhibited low levels of heterosis for these traits, interspecific diploids produced moderate levels of heterosis in greenhouse experiments. Differences between greenhouse and field trial results can largely be explained by pest damage, which negatively impacted interspecific diploids. Heterosis for the traits that form the basis for biomass yield, including stem growth, foliar, and physiological traits, was quantified and family-level differences are discussed.