Leptosphaeria maculans, which causes blackleg (phoma stem canker) on canola (oilseed rape), is an important pathogen worldwide. To assist in standardising inoculation procedures for resistance screening, studies were conducted on the conditions that influence germination of ascospores. Germination of, and hyphal growth from, ascospores of L. maculans were studied at 5–20°C on agar-coated slides, detached leaves and cotyledons of five canola cultivars in South Australia. Germination began after 2 h at 10–20°C and 4 h at 5°C. After 24 h, the percentage germination of ascospores was greater at the higher temperatures of 15 and 20°C, compared with the lower temperatures of 5 and 10°C. This optimum temperature range was similar for ascospores on agar and on plant tissue. Percentage ascospore germination was greater on agar-coated slides and cotyledons than on leaves for all cultivars tested. Germination was greater on leaves or cotyledons of the susceptible cultivars, Q2 and Karoo, than on the resistant cultivars, Hyola 60 and Ripper. Elongation of germ tubes was generally greater on cotyledons than on leaves of the cultivars tested and also greater on susceptible cultivars than resistant cultivars after 24 h at 15–20°C. However, at 5°C there was no difference in the mean length of germ tubes between leaves and cotyledons of each of the five cultivars. In summary, temperature, cultivar and plant tissue significantly affected germination of, and hyphal growth from, ascospores in the controlled-environment conditions.