Detecting pathogens in asymptomatic plants allows for a rapid response to emerging threats, such as the introduction of new pathogens or the emergence of new resistant strains. Frosty pod rot (Moniliophthora roreri) significantly jeopardizes cacao production by impairing pod quality and yield. However, early diagnosis is challenging due to its prolonged incubation period, compounded by symptoms resembling those of M. perniciosa, the causative agent of witches' broom disease. To address this, a specific and sensitive quantitative real-time PCR (qPCR) assay was developed for M. roreri detection. This involved testing diagnostic primers against a panel of 252 DNA samples collected from target and nontarget species of over 13 fungal species commonly found in cacao plantations from various regions. Species-specific primer pairs were obtained through genomic comparison of Moniliophthora spp. genomes. Two primer sets, Mr77F/R and Mr78F/R, were validated using a set of M. roreri DNA samples representative of different genetic groups (Ecuador, Peru, and Mexico) and M. perniciosa, from which no amplification was detected. The limit of detection (LOD) was determined to be 0.9 ng/µl for both primers Mr77F/R and Mr78F/R. Using this set of primers, the amplification of M. roreri DNA from naturally diseased cocoa pods at various stages of infection was also successful. At the practical level, the data provided here confirm the value of molecular diagnostic testing for the early detection of M. roreri. These features are desirable for improving fungal diagnostic capacity and assisting in devising strategies to avoid pathogen dispersion within cacao-growing regions.