Two different inoculation techniques were investigated before studying the reaction of the major rust resistance gene of sugarcane cultivar R 570 against isolates of Puccinia melanocephala from different geographic locations. Cultivar R 570 exhibited severe rust symptoms when in vitro plantlets were inoculated with a rust isolate from Réunion Island, but a good correlation with field resistance was observed when detached leaves were inoculated with the pathogen. This latter technique was then used to inoculate R 570 and a sample of its self progeny with rust isolates from Brazil, Colombia, Florida (three isolates), Guadeloupe, Réunion Island, and Zimbabwe. R 570 was resistant to all isolates of P. melanocephala, and the segregation of resistance in the progeny did not change with the isolates, suggesting that a single gene, or a single chromosomic region, was involved in the resistance against all tested isolates. This major resistance gene has, therefore, potential value to improve resistance to rust in various geographic regions.