Soybean (Glycine max) acreage is increasing dramatically, together with the use of soybean as a source of vegetable protein and oil. However, soybean production is affected by several diseases, especially diseases caused by fungal seed-borne pathogens. As infected seeds often appear symptomless, diagnosis by applying accurate detection techniques is essential to prevent propagation of pathogens. Seed incubation on culture media is the traditional method to detect such pathogens. This method is simple, but fungi have to develop axenically and expert mycologists are required for species identification. Even experts may not be able to provide reliable type level identification because of close similarities between species. Other pathogens are soil-borne. Here, traditional methods for detection and identification pose even greater problems. Recently, molecular methods, based on analyzing DNA, have been developed for sensitive and specific identification. Here, we provide an overview of available molecular assays to identify species of the generaDiaporthe,Sclerotinia,Colletotrichum,Fusarium,Cercospora,Septoria,Macrophomina,Phialophora,Rhizoctonia,Phakopsora,Phytophthora, andPythium,causing soybean diseases. We also describe the basic steps in establishing PCR-based detection methods, and we discuss potentials and challenges in using such assays.