Totally, 1.5 million fungal species estimated, of which less than 10 percent have been described to date. Recent advances in molecular biology as well as gene sequencing technologies facilitate the discovery and identification of new species in various groups of living organisms, especially fungi. Morphological methods do not have efficiency for the identification of fungal species, so, using new techniques based on DNA data has been considered in the rapid and accurate fungal species identification. DNA Barcoding is a new technique with several years’ backgrounds for detecting fungi and fungal like organisms. The Internal Transcribed Spacer in ribosomal RNA gene (ITS-rDNA) has been considered as a suitable barcode sequence. The efficacy of this region’s sequence data in most fungal groups has led to its use as a standard barcode by different mycologists. However, the ITS-rDNA region appears to be used as the primary barcode sequence for fungi of the genus or species group. The accurate species-specific identification is performed depending on the fungal group based on secondary barcodes. Due to the pleomorphic nature of fungi, the use of DNA barcoding is of importance. To date, hundreds of thousands of reference barcode sequences have been created for thousands of species of living organisms through DNA barcoding projects. The subsequent efforts of scientists are managing and automation of DNA barcoding in order to speed up the identification process.