An incursion of sugarcane smut in Queensland was identified in 2006 in Bundaberg, Ingham, and Mackay. In 2008, two cultivars, ‘Q205’ and ‘Q170’, were highly susceptible in Bundaberg but remained disease free on a heavily infested farm in Mackay. A glasshouse experiment was established to determine whether the differences in disease expression were due to the sources of the two cultivars sourced from Bundaberg and Mackay in 2009. These were inoculated with the Mackay population of Sporisorium scitamineum spores. These cultivars had the same levels of disease, indicating that the variation in the clonal source of the cultivars was not responsible for the observed differences. A second glasshouse experiment in 2013 confirmed that highly susceptible Q205 and Q170 were resistant to the Mackay population of S. scitamineum but remained susceptible to the Bundaberg population. In 2010, S. scitamineum populations of smut fungi from Mackay and Bundaberg were compared in a field trial and the Mackay population had significantly less disease than the Bundaberg population on four cultivars (Q170, Q205, ‘Q174’, and ‘Q138’) but had significantly more disease in ‘Q188’. These results confirmed the field observations and suggested that there was a differential response among the cultivars for the smut fungi collected from Mackay compared with Bundaberg and are genetically different. This finding suggests that a mixture of spores should be used to inoculate sugarcane clones for resistance screening.