The sustainability of purpose-grown biomass production on marginal lands in Canada is uncertain. In this study, an assessment of biomass yield and sustainability was performed for two poplar clones (Poplus deltoides × P. nigra, DN-34—PDN, and P. nigra × P. maximowiczii, NM-6—PNM) and two switchgrass cultivars (Panicum virgatum ‘Cave-in-Rock’—SGC, and P. virgatum ‘Nebraska’—SGN) on three marginal lands in Guelph (ON), Kemptville (ON), and Nappan (NS) in Canada. The differences in stem biomass across sites were not significant; however, differences in stem biomass among plants were statistically significant between poplar and switchgrass (p < 0.0001) and between poplar clones (p < 0.0001). The 2-yr stem biomass yield in PNM (15.27 ± 1.28 t ha^-1) was significantly higher than those in PDN (7.02 ± 0.54 t ha^-1), SGC (2.57 ± 0.28 t ha^-1), and SGN (1.45 ± 0.22 t ha^-1). Two sustainability indices based on macronutrients (MBSI) and nitrogen (NBSI), were developed to assess sustainability. Both indices show that the biomass production system of high-yielding poplar clone PNM depicts nutrient loss and may require external nutrient inputs via fertilization during the establishment phase. Higher index values for switchgrass SGC (1.47 ± 0.22, 1.11 ± 0.15) and SGN (1.37 ± 0.16, 1.17 ± 0.12) for MBSI and NBSI, respectively, indicate that despite low stem biomass yields, switchgrass biomass production is sustainable. These findings suggest that, from a nutrient perspective, sustainable biomass production systems can be established on marginal lands in Canada; however, there is a trade-off between high yield and long-term sustainability in purpose-grown biomass production systems.