In the province of Ontario, Canada, markets are emerging for biomass for a range of end-uses including combustion, gasification and bio-products. Given concerns over the technical feasibility and sustainability of crop residue removal, dedicated C4 perennial grasses were identified as potential candidates to meet this emerging demand. In 2008, a multi-site trial was initiated across the province to comparatively evaluate C4 perennial grasses, including miscanthus (Miscanthus spp.), switchgrass (Panicum virgatum) and big bluestem (Andropogon gerardii). Four varieties of miscanthus (M. sinensis × M. sacchariflorus - Nagara, Amuri, M1 Select and Polish) were compared to two upland varieties of switchgrass (Cave-in-Rock and Shelter), two varieties of big bluestem (Prairieview and Southlow), and one variety of prairie cordgrass (Red River). Treatments were set up in a factorial experiment with four nitrogen rates (0, 40, 80, 160 kg N ha-1) and two harvest dates (late fall and early spring). A treatment representing the prominent land use pattern in the region was also included to assess land use change effects and relative biomass yield. Data is given for measurements taken to assess treatment effects on establishment success, winter survival, yield and moisture content. Variation in winter tolerance was observed, but it can be concluded that varieties for each of the evaluated species exist that are adapted and suitable to be grown under Ontario conditions. For many of the measured parameters, including yield, significant interactions between year, location, species, variety, and agronomic management were observed.