Panama disease, caused by Fusarium oxysporum f. sp. cubense (Foc), is ranked among the most destructive disease of banana. The use of resistant varieties is the most desirable and effective control measure. Since durability of the resistance and effective cultivar deployment is strongly linked to pathogen population structure, information on this regard is essential. In this study, 214 Foc isolates from different banana producing regions of Brazil [Northeastern, Southeastern and Southern] were analyzed. Initially, nine microsatellite markers (SSR) were tested, which revealed 52 distinct haplotypes distributed in the different geographical regions and cultivars. While AMOVA analysis showed that 68.01% of the total variation occurred within the states, correlation between genetic and geographical distances was only found in the Southern region. Results indicated that isolates from different states comprise a single population, which is predominantly clonal. When isolates representing different haplotypes were inoculated in four banana cultivars, differences in severity were found, and the high severity values were caused by isolates from haplotypes H7, H41 and H31. The diversity found points to the need for additional studies since this characteristic may be related to Foc's evolutionary potential and possibly to its ability to overcome the resistance from breeding program-generated cultivars. This is the most comprehensive study on population biology of Foc in Brazil.This article is protected by copyright. All rights reserved.