Soybean (Glycine max L.) is one of the most economically important crops in Uruguay. Soybean is affected by several pathogens, including fungal Diaporthe species that cause soybean stem canker (SSC), which reduces yield worldwide. The main pathogen causing SSC are Diaporthe aspalathi, D. caulivora, D. masirevicii, D. miriciae and D. longicolla (Mena et al. 2020; Mena et al. 2024). Disease symptoms are similar between Diaporthe species and consist in brown-to-reddish necrotic lesions on the stems. Similar morphological and disease characteristics of these pathogens constitutes a challenge for conventional disease diagnosis. The present study was carried out to detect and quantify Diaporthe species associated to SSC in Uruguay by multiplex qPCR. Four species-specific TaqMan primer-probe sets were designed based on translation elongation factor 1-alpha gene (tEF1a) sequences for the species: D. aspalathi, D. caulivora, D. miriciae and D. longicolla. The specificity and efficiency of the primer-probe sets were tested using PCR products and genomic DNA from pure cultures of Diaporthe species. In addition, multiplex qPCR assay was evaluated in soybean plants inoculated with one or more Diaporthe species. Our results indicate that these Diaporthe species can be detected and quantified alone and in parallel with a multiplex qPCR assay. Thus, our qPCR assay could be a useful tool for diagnosis of D. aspalathi, D. caulivora, D. miriciae and D. longicolla, as well as for designing strategies to manage SSC disease.