Fungi have diverse associations with plants, encompassing over a million species of endophytes, mycorrhiza, pathogens, and saprophytes. This varied community of fungi play a significant role in plant growth and health particularly by suppressing pathogens. Prompt and error-free techniques of detection and identification of fungi inhabiting plants are essential for their applications in a variety of sectors as well as controlling fungal diseases. Conventional methods such as visual analysis of symptoms, culturing, and isozyme analysis depending upon phenotypic and biochemical characters are rapid and economically viable; however, they require more time and are less sensitive. Nucleic acid-based techniques have evolved as a faster and better alternative providing accurate characterization and differentiation of plant associated fungi by detecting specific target nucleic acids. It involves genomic DNA extraction by efficiently lysing the fungal cells followed by its purification and quantification. Currently real-time polymerase chain reaction (RT-PCR) widely known as quantitative PCR (qPCR) is being used to detect fungi inhabiting various plant parts. The following chapter describes the protocol of RT-PCR-mediated detection of plant-associated fungi.