Pseudocercospora musae, causal agent of Sigatoka leaf spot, or yellow Sigatoka disease, is considered a major pathogen of banana (Musa spp.). Widely disseminated in Brazil, this study explored the genetic diversity in field populations of the pathogen from production areas in the Distrito Federal and the States of Bahia, Minas Gerais, and Rio Grande do Norte. Resistance to demethylation inhibitor (DMI) fungicides was also examined. For 162 isolates from 10 banana growing regions, analysis of mating type idiomorph frequency was conducted, together with estimation of genetic diversity at 15 microsatellite loci. A total of 149 haplotypes were identified across the examined populations, with an average genetic diversity of 4.06. In general, populations displayed 1:1 proportions of idiomorphs MAT1-1 and MAT1-2, providing evidence for sexual recombination. Multilocus linkage disequilibrium also indicated asexual reproduction contributing to the genetic structure of certain populations. AMOVA revealed that 86.3% of the genetic differentiation of the pathogen occurred among isolates within populations. Discriminant Analysis of Principal Components (DAPC) identified six most probable genetic groups, with no population structure associated with geographic origin or collection site. Although genetic similarity was observed among certain populations from different states, data revealed increasing genetic differentiation with increasing geographic distance, as validated by Mantel's test (r = 0.19, P < 0.001). On the basis of DMI fungicide sensitivity testing and CYP51 gene sequence polymorphism, isolates from the Distrito Federal separated into two main groups, one with generally higher EC50 values against eight DMI fungicides. A clear phenotype-to-genotype relationship was observed for isolates carrying the CYP51 alteration Y461N. Conventionally adopted fungicides for control of Sigatoka leaf spot are likely to be overcome by combined sexual and asexual reproduction mechanisms in P. musae driving genetic variability. Continued analysis of pathogen genetic diversity and monitoring of DMI sensitivity profiles of Brazilian field populations is essential for the development of integrated control strategies based on host resistance breeding and rational design of fungicide regimes.