Orange rust of sugarcane caused by Puccinia kuehnii was first reported in Florida in 2007. Since then, several sugarcane cultivars that were resistant during the initial epidemics became susceptible within a few years. These shifts in resistance were attributed to the evolution of the pathogen and appearance of new races. To study the variation in virulence of P. kuehnii, healthy leaf pieces of sugarcane cultivars susceptible to orange rust were brush inoculated with isolates of P. kuehnii collected from susceptible cultivars in the field. After inoculation, leaf pieces were placed in an incubator and disease severity based on the number of rust uredinia was determined 2 weeks postinoculation. Isolates of P. kuehnii collected from sugarcane cultivar CP 89-2143, which only showed severe symptoms of orange rust starting in 2011–2012, produced 300%–500% more uredinia on CP 89-2143 than the isolates collected from cultivar CL 85-1040 that has been susceptible since 2007. Sugarcane cultivar CL 85-1040 exhibited high and equivalent numbers of uredinia regardless of the inoculated isolate of the pathogen. These data support the occurrence of pathogenic specialization within P. kuehnii and the existence of at least two races of this pathogen in Florida. Analysis of amplified fragment-length polymorphism among isolates of P. kuehnii from cultivars CP 89-2143 and CL 85-1040 differing in resistance to orange rust revealed genetic variation among rust uredinia. However, this variation was not associated with a specific sugarcane cultivar, suggesting that pathogenic variation was not linked to major, but rather to small genetic changes within the genome of P. kuehnii.