Soybean (Glycine max) is one of the most economically important crops in Uruguay and is affected by soybean stem canker (SSC) disease. The main pathogens involved are Diaporthe aspalathi, D. caulivora, D. longicolla and D. miriciae, causing similar symptoms, including brown-to-reddish necrotic lesions on stems and wrinkled, cracked seed coats. The presence of these pathogens in asymptomatic plants, along with their similar morphology, constitutes a challenge for conventional disease diagnosis. Currently, no resistant cultivars are available for all Diaporthe species, and effective control methods are lacking, making prevention the primary strategy. Therefore, early detection and accurate identification of the specific Diaporthe species involved are essential for minimising losses. This study aimed to detect and quantify the three most prevalent Diaporthe species in Uruguay, D. caulivora, D. longicolla and D. miriciae, and identify D. aspalathi in stem canker lesions using multiplex quantitative PCR (qPCR). Species-specific TaqMan primer-probe sets targeting the translation elongation factor 1-α gene (tEF1a) were designed for each Diaporthe spp. The specificity and sensitivity of the primer-probe sets were tested using genomic DNA. The assay was validated using stem tissues and seed samples artificially inoculated with one or more Diaporthe species. Additionally, the multiplex qPCR assay was evaluated in field-collected soybean stem and seed samples in 2024, and seed lots from different origins. Our results demonstrate that this multiplex qPCR assay effectively detects and quantifies Diaporthe species individually and simultaneously, providing a valuable diagnostic tool for seed certification, pathogen surveillance, and the development of effective strategies for SSC management.