Sustainable, renewable energy production from lignocellulosic bioenergy crops such as shrub willow (Salix spp.), can help offset fossil fuel usage without competing with food crops. Hybridization is a key component in shrub willow breeding because hybrids often exhibit heterosis for yield. The genomic basis of heterosis in willow has not been well characterized, especially in high-yielding triploid hybrids. The primary objectives of this study were 1) to evaluate diverse diploid, triploid, and tetraploid families for a suite of traits important for biomass production, 2) to identify genomic regions linked to these traits, 3) to investigate the patterns gene expression in diploid and triploid hybrids, and 4) to correlate heterosis with gene expression in triploid hybrids. This work supports evidence that triploid hybrids of shrub willow exhibit heterosis for biomass growth traits. A high proportion of expression-level dominance and additive inheritance was observed among triploids, as well as cis- and trans-regulatory divergence. Importantly, nonadditive gene expression correlates with heterosis for biomass yield and growth traits among triploid hybrids. The extensive phenotypes collected will provide a database for future trait mapping and serve as a foundation for genomic selection. These results offer a unique perspective on the genomic basis of heterosis in high-yielding triploid hybrids, and will contribute to the growing genomic toolkit for the improvement of shrub willow as a bioenergy crop.