Brown rust, caused by Puccinia melanocephala, is an important disease of sugarcane worldwide, controlled primarily with host plant resistance. Disease response shifts from resistant to susceptible have been repeatedly observed for cultivars. However, information is limited concerning pathogen variability related to host reactions. To evaluate variability in the pathogen population and characterize resistance responses in different host genotypes, seven cultivars were inoculated with four urediniospore collections from three cultivars. Greenhouse-grown plants were inoculated under controlled conditions favorable for infection and disease development. Severity assessed as leaf area occupied by lesions, lesion density, and lesion size was determined and compared. Three cultivars that shifted from resistance to high susceptibility while under cultivation exhibited differential disease severity when inoculated with spore collections from two of the respective cultivars. Two cultivars exhibited consistent moderate to high levels of quantitative resistance against all spore collections and two cultivars, including one with the Bru1 resistance gene, were highly resistant to all collections. Differential reactions were best revealed by assessing percent leaf area. Pathogenic variability related to host genotype was confirmed, and quantitative resistance was detected that could be useful to improve breeding and selection for effective, durable resistance to brown rust.