Willow biomass is used as a bioenergy source in various conversion technologies. It is noteworthy that apart from the beneficial environmental impact of a willow plantation, the biomass quality is also very important as it has an impact on the effectiveness of its use and emissions produced in various bioenergy technologies. Therefore, this study analysed the thermophysical properties and elemental composition of 15 genotypes of willow biomass from two plantations situated in the north of Poland, harvested in two consecutive three-year rotations. The differences in the moisture content, ash content and the lower heating value were mainly determined by the genotype, i.e., by genetic factors. In contrast, the content of carbon, nitrogen, sulphur and hydrogen was determined by the location (environmental factors), but also by the genotype, and by a combination of these factors. The following were the mean levels of the willow biomass characteristics, regardless of the location, genotype and harvest rotation: 48.9% moisture content, 1.26% d.m. ash content, 19.4% d.m. fixed carbon, 79.4% d.m. volatile matter, 19.53 MJ kg−1 d.m. higher heating value, 8.20 MJ kg−1 lower heating value, 52.90% d.m. carbon, 6.23% d.m. hydrogen, 0.032% d.m. sulphur, 0.42% d.m. nitrogen. The present research has shown that the selection of the willow genotype is important for the quality of biomass as energy feedstock. However, plantation location, as well as successive harvest rotations, can have a significant impact on the biomass elemental composition.