Tiger Milk Mushroom (Lignosus spp.) specimens obtained from field collection usually consist of only the sclerotia, without intact cap and/or stipe making species identification an arduous task. In this chapter, we describe the DNA barcode marker approach based on the internal transcribed spacer region via polymerase chain reaction technology to enable rapid and precise identification of species found in this genus. DNA markers are highly specific and easily amplified, allowing rapid identification and reliable authentication of Lignosus species.