The lichens Tephromela atra and Tephromela grumosa were studied in detail using various techniques, including morphological identification, biochemical taxonomy (chemotaxonomy), anatomical, and molecular characterization. The molecular characterization of Tephromela atra and Tephromela grumosa (Pers.) Hafelner & Cl. Roux was analyzed using internal transcribed spacer rDNA (ITS1) genes and the sequences obtained were compared with the ITS sequences of the other Tephromela species in GenBank (NCBI). The sequences were analyzed against reference sequences from the GenBank databases using the programs; BLAST, ClustalW, and MEGA11. The phylogenetic relationship was evaluated. The results show that some samples of Tephromela atra obtained in this study are closer to the same species isolated from Greece, while other samples are closer to other samples isolated from Italy and Austria. Moreover, the Tephromela grumosa isolated from Eskisehir Bozdag Mountain form a separate branch closer to the Tephromela grumosa samples isolated from Italy. Our study shows that lichens are an important source of biodiversity in Turkiye, which has a high species richness in Anatolia.