The witches' broom (Moniliophthora perniciosa) is considered as one of the main threats for cacao production and, consequently, for chocolate production worldwide. In this work, the genetic diversity and population structure of M. perniciosa were analyzed for 59 isolates collected in five departments of Colombia and using 10 microsatellite markers. Analyses revealed 35 multilocus genotypes and clonal populations structure according to linkage disequilibrium analysis. One of the objectives of this study was to determine whether populations were differentiated by geographic origin or Theobroma cacao host genotype. Analysis of molecular variance, discriminant analysis of principal components, and Bruvo genetic distance suggested that the genetic structure was driven by geographic origin and not by T. cacao genotype. The results of this study were consistent with previous findings obtained in other cocoa-producing countries. Important insights were discussed regarding the dispersal patterns of the pathogen in Colombia and the genetic change of its populations because of different environmental conditions.