Brown Rust (BR) is one the most important diseases of sugarcane worldwide, causing yield losses between 10 to 40% in susceptible varieties, requiring breeding towards resistance. Considering pathogen´s coevolution, planted area and environmental factors, BR overcame resistance in several varieties at the Cauca valley in Colombia, indicating progressive adaptation. To address this issue, this work standardized BR inoculations on 2-3 months old plants at semi-controlled conditions, and determined morphology and pathogenicity of 15 single pustule BR pure isolates from four varieties with economic and breeding importance to Colombia´s industry. The 15 isolates were cross- inoculated on 11 sugarcane varieties, contrasting in origin, genetic background and BR response. Morphometric characterization of urediniospores and paraphyses showed significant differences between isolates and found relationships with BR severity. Cross inoculations showed statistical differences between isolates and within isolates obtained from the same variety, with high variation in severity, incubation and latent periods, and reaction or disease phenotype. There was not relationship between isolate origin and severity on the variety source. Based on severity and reaction, varieties Pindar, Hinds Special and Q-117 showed differential responses. Results supported BR adaptation through the historical transition of commercial varieties MZC 74-275, CP 57-603, CC 84-75 and CC 85-92 in the Cauca valley, providing also elements to study pathogen diversity and incomplete resistance to BR in sugarcane varieties.