Moniliophthora perniciosa presents discrepancies in its taxonomy and is the causal agent of cocoa witches' broom, which in Colombia is the second disease after moniliasis (Moniliophthora roreri). In this study 12 samples of the pathogen obtained from different cocoa materials from the Casa Luker S.A. Farm (Palestina, Caldas, Colombia) were analyzed. The DNA was obtained from monosporic cultures and from the fungus fructification. Each sample was evaluated from microbiological cultures using molecular markers ITS (ITS1, ITS2 and 5.8S genes). The Molecular ADN characterization obtained from direct extraction from the fungus fructification avoids artificial selection in the culture means and ensures a more realistic diagnosis of the pathogen diversity in the plantations. The molecular characterization allowed differentiation between M. pernicious and M. roreri.