Moniliophthora perniciosa, the founder agent of the ‘witch’s broom’ on cocoa (Theobroma cacao L.) is one of the most important diseases in cocoa plantations, causing economic losses close to 70% worldwide and 40% nationwide. It shows a high genetic variability and discrepancies in its taxonomy. Characterization of the genetic diversity of biotypes is important for projects aimed towards the handling of this pathogen and the development of resistant cocoa materials. Twelve isolations of the fungus were analyzed in this study from different cocoa material. Each sample was evaluated with molecular markers directed towards a nuclear ribosomal DNA (rDNA) region known as ITS (Internal Transcribed Spacer), an intergenic region (IGS-1), and five simple sequence repeats (SSR). The IGS-1 allowed the determination of biotype C, however, an evident genetic variability was found within this biotype that has not been yet reported. The genetic diversity analysis of M. perniciosa by microsatellite markers gave a total value of 0.4260, a total heterozygosity of 0.6143, and a polymorphism information content (PIC) of 0.3407; these values are considered to be within a medium to high range for the studied isolations, and are an estimation of the genetic variability present in M. perniciosa.