Weeds are a major constraint in canola (Brassica napus L.) production worldwide, as they cause significant reductions in seed yield and quality. Crop interference is one of the approaches to tackle weed infestation along with other agronomic interventions. In Australia, studies have shown genetic variation in the canola capability to suppress annual ryegrass (Lolium rigidum Gaudin) in the field and under in vitro conditions. Early-season crop biomass accumulation and greater plant height are desired attributes for suppression weeds in canola. However, the canola ideotype for interference traits against this weed has not been studied under glasshouse conditions. In this study, we compared the competitive ability of 26 canola genotypes against annual ryegrass under both glasshouse and field conditions. Five canola genotypes consistently showed the ability to suppress growth of annual ryegrass. Both at glasshouse and field conditions, the shoot biomass, largely contributed by leaf biomass, was significantly associated with suppression ability. Our results suggest that a glasshouse-based evaluation approach can be used to determine the suppressive ability of advanced breeding lines for suppression of ryegrass growth. Based on our analysis, we suggest that initial screening of large collections of germplasm can be conducted under glasshouse conditions, with selected genotypes further evaluated in the field.