Since the first outbreak of orange rust in Brazil in 2019, resistant sugarcane cultivars were used to control this disease caused by Puccinia kuehnii. Nevertheless, new rust epidemics occurred on cultivars previously considered resistant, suggesting variation in virulence of the pathogen. Variability in virulence of P. kuehnii was investigated herein using three different inoculum sources of P. kuehnii from Brazil. In an initial screening, 1,068 single-uredinium rust isolates from three susceptible cultivars were each inoculated in the laboratory on one of five sugarcane cultivars resistant to orange rust. A five-cm leaf section was brush-inoculated with all spores of each uredinium and plants were maintained in a humid chamber for 24 hours before transfer to a shed. The size of leaf areas with disease lesions was measured 21 days after inoculation with image analysis software Assess 2.0. Fourteen isolates of the pathogen produced uredinia on a leaf of resistant cultivars. Capacity of infecting a resistant cultivar was confirmed for one of these variants in replicated experiments with spores from previously inoculated leaves. Uredinia of P. kuehnii from sugarcane cultivar SP89-1115 that developed on leaves of three resistant cultivars were differentiated from uredinia infecting only a susceptible cultivar by microsatellite marker NPRL_PK_221_spka. Populations of P. kuehnii infecting susceptible cultivars therefore include sub-populations that could be the initial inoculum for development of new epidemics on cultivars resistant to orange rust.