The importance of fungal species differentiation is critical for plant pathology research and disease control purposes. Traditionally, fungal species have been identified by a range of cultural and morphological characteristics, such as conidial morphology, presence or absence of setae, fungicide sensitivity, and colony color and growth rate. Although valuable, these criteria alone are not always adequate as morphological characteristics may vary under different environmental conditions. Among other techniques, sequence analyses of the internal transcribed spacer (ITS) regions, including the 5.8S rDNA, has been used extensively to identify fungal species. This region is the most widely sequenced DNA region of fungi and has been employed as the universal fungal barcode sequence. It is very useful for molecular systematics at species to genus level and within species.This protocol is useful for a primary molecular and accurate identification of any fungal species using the sequence analyses of the ITS-5.8S rDNA region