Despite the importance of coffee leaf rust (Hemileia vastatrix), population studies of the pathogen are mostly focussed on identification of physiological races and there is no information on the genetic diversity and structure of populations of H. vastatrix in Brazil. In this study, 120 isolates of H. vastatrix were collected from organic and conventional coffee production systems from two coffee-producing regions in the state of Minas Gerais, Brazil. To test the hypothesis that H. vastatrix populations differ between conventional and organic plantations, random amplified polymorphic DNA markers were used to determine the genetic diversity and structure of the populations of H. vastatrix sampled from 12 fields within the two regions. Across all loci, high values of genetic diversity (h = 0.28) and 92 haplotypes were detected. Based on the cluster analysis and θ test, there was no association between origin of the isolate with either cropping system or region. In addition, considering the AMOVA analysis, most of the genetic variance occurred within populations (≈80%). There is evidence of random mating in the population of H. vastatrix in Brazil, which can partially explain the high genetic variability of the pathogen. Therefore, we conclude that genetic diversity in H. vastatrix populations in Minas Gerais was high and no clear clustering of isolates was detected that allowed establishment of correlation of pathogen diversity with geographical origin or coffee cropping system.