Soybean stem canker (SSC) is an important disease which reduces yield worldwide. SSC is caused by Diaporthe caulivora, D. aspalathi and D. longicolla. Symptoms produced by Diaporthe species are similar, with brown-to-reddish necrotic lesions on the stems. Fifty-three isolates of Diaporthe were isolated from soybean stem samples with symptoms of SSC in Uruguayan fields during 2022-2023. In this study we identified D. caulivora, D. longicolla, D. miriciae and D. masirevicii by secuencing the internal transcribed spacer (ITS) region and translation elongation factor-1-alpha gene (tEF1a). Morphology of D. miriciae and D. masirevicii isolates was observed. Inoculations of D. miriciae D67 and D. masirevicii D99 were done in soybeans susceptible plants cv.Williams. Dark brown, necrotic lesions were observed on inoculated soybean stems at 11 dpi, which were similar to symptoms of SSC observed in the field. At 11 dpi pynidia appeared on the soybean stem around the wound. To our knowledge, this is the first report of D. masirevicii causing SSC worldwide and the first report of D. miriciae as a pathogen causing SSC in Uruguay. Due to the economic importance of this disease elsewhere, further research on Diaporthe species, pathogenicity and effective detection methods are warranted to mitigate potential crop losses.