Macrofungi belonging to the phylum Basidiomycota are mostly used as medicinal mushrooms in many countries. In the present study, hundred basidiocarp of macrofungi were collected from Tamilnadu during rainy season. The basidiocarp was found in association with root/trunk of living trees, wood log and decayed matter. Among the hundred basidiocarp, 49 were grown into axenic cultures. Notable variations in the macroscopic characteristics of the basidiome and culture morphology were observed. To study the genetic diversity, the molecular taxonomy of the isolates was carried out using internal transcribed spacer (ITS) and 5.8S rRNA gene sequence marker. Thirty-two strains belonging to the order Polyporales, Hymenochataeles and Russuales under the division Basidiomycota were classified based on phylogeny analysis. This study provides first evidence for the occurrence of species Fulvifomes fastuosus (LDCMY39 and LDCMY43) and Ganoderma wiiroense (LDCMY02, LDCMY08, LDCMY11, LDCMY17 and LDCMY19) from southern India. Molecular evidence for the existence of Phellinus badius was given for the first time as well. These data enhance our understanding on the diversity of macrofungi in India, which could be further exploited for biomedical applications.