Moniliophthora perniciosa is a hemibiotrophic plant pathogen that causes witches' broom disease (WBD) in Theobroma, Herrania, and other plant species. This disease is one of the primary threats that reduce the yield of cacao and cupuaçu plantations in Tropical America. Thereby, there is a growing concern that the pathogen may spread to other areas. The fungus can infect flowers, pods, and stems, causing various symptoms, including stem swellings and dark brown necrotic pods. In some cases, these symptomatologies are very similar to other cacao diseases, such as the black pod (Phytophthora spp.) and frosty pod rot (M. roreri) of cacao. Pathogens isolation and identification from diseased samples need expertise. Thus, the objective of this work was to develop a diagnostic guide with standard procedures with the aid of a molecular marker for M. perniciosa identification that is specific, sensitive, and cost-effective. A genomics-based approach was used to identify unique primer pairs to M. perniciosa. The specificity of 24 primer pairs in identifying M. perniciosa from pure culture gDNA was evaluated by conventional polymerase chain reaction (PCR) with approximately 400 isolates from ten fungal species of common occurrence in cacao plantations, including M. roreri. No amplicon was detected from other fungal species, confirming the high specificity and sensibility of the M. perniciosa specific markers Mp44 and Mp47 for molecular identification of M. perniciosa from pure culture. Finally, the present study provides a set of standardized WBD diagnosis procedures that associate a classical with a molecular approach by PCR-based identification of M. perniciosa.