Aims The allelopathic activity of canola (Brassica napus) germplasm was investigated using the important Australian weed, annual ryegrass (Lolium rigidum) as the target species. Methods Three different canola plant densities (10, 20, and 30 seedlings/beaker) of each of 70 world-wide genotypes were tested in vitro in close proximity to annual ryegrass seedlings.Results The allelopathic activity of canola, as measured by reduction in annual ryegrass root and shoot growth, increased with canola crop seedling densities. Density did not consistently influence shoot length of annual ryegrass. Greater shoot length suppression was observed in genotype cv. Rivette and BLN3343C00402. The Australian genotype cv. Av-opal and the breeding line Pak85388-502 suppressed root length of ryegrass more than other genotypes, even at low densities. At the lowest density, the least allelopathic genotypes were cv. Barossa and cv. Cescaljarni-repka, although they became more allelopathic at higher density. An overall inhibition index was calculated to rank each of the canola genotypes. There were significant differences between canola genotypes in their ability to inhibit root and shoot growth in ryegrass. Conclusion Considerable genetic variation exists among canola genotypes for their allelopathic effects on annual ryegrass. Further investigation is required to determine the allelopathic mechanisms, particularly to identify the responsible allelochemical(s) and the gene(s) controlling the trait. This research suggests that highly allelopathic canola genotypes can be potential for controlling weeds such as annual ryegrass in integrated weed management programs.