No sugarcane cultivar is resistant to root-lesion (Pratylenchus zeae) nematode in Australia. Sugar Research Australia commenced a research project to evaluate new sets of accession lines derived from introgression breeding between wild relatives of sugarcane and commercial parents. No established method of assessment was available for comparing the resistance of sugarcane in the glasshouse. This study aimed to determine the most suitable assessment method for comparing resistance in accession lines. Two resistance parameters were measured, (i) number of nematodes per plant, and (ii) number of nematodes per g of roots. Nine screening trials were conducted from 2011 to 2020. Resistance parameter number of nematodes/plant had less variations within trials compared to nematodes/g roots, although both parameters were equally repeatable. Number of nematodes/g of root were negatively correlated (r = -0.29 to -0.86, p ≤ 0.001) with root biomass in all nine trials, and with shoot biomass in three out of seven trials (r = -0.23 to -0.31, p ≤ 0.05). In contrast, the number of nematodes per plant were positively correlated with shoot biomass (r = 0.25-0.53, p ≤ 0.05) in three out of seven trials and with root biomass (r = 0.17-0.27, p ≤ 00.05) in three out of nine trials. These results clearly indicated that nematodes per g root is related to reduction in root biomass and shoot biomass.