Early diagnosis of seedborne fungal pathogens is particularly important, as often, infected seeds appear symptomless; seed diagnosis can avoid uncontrolled propagation of pathogens through long-distance exchange of such material. This will prevent economic losses and unnecessary use of fungicides, so reducing costs and the introduction of toxic substances into the environment. Traditional techniques for detection of seedborne fungi are based on incubation and grow-out methods. Although these are frequently used because of their simplicity of application, they are time-consuming, require mycological skills, and are sometimes not sensitive enough to low levels of seed infection. Recently, new identification techniques, based on DNA analysis, have been applied and are very efficient due to high sensitivity and specificity. The most common technique is conventional PCR, while other recent techniques include nested PCR, to obviate low levels of target pathogens, multiplex PCR, to detect several pathogens simultaneously, real-time PCR, to quantify fungi on seeds, and magnetic-capture hybridization PCR. The main drawbacks of molecular methods are the inability to distinguish between vital and non-vital inocula, and the difficulty in obtaining quality DNA template, due to PCR inhibitors in seeds. To reduce inhibitory effects, several modified PCR protocols, such as loop-mediated isothermal amplification, and non-destructive testing methods have been developed. Loop-mediated isothermal amplification and next-generation sequencing have been widely applied in nucleic acid analysis and, given the numerous advantages provided, their application can be substantially extended in the future for detection of fungal pathogens in seeds.