Proper species recognition is required to correctly estimate species preferences and their vulnerability or for eco-evolutionary inference. Varicellaria hemisphaerica and Varicellaria lactea are almost completely morphologically homogeneous species with unclear identification features. To evaluate the importance of morphological, chemical, and ecological characteristics used in recognition of these species, we tested 670 specimens, of which 42 were analyzed phylogenetically using nucITS rDNA, SSU rDNA, and LSU rDNA markers. This integrated taxonomical approach showed that V. hemisphaerica is distinct from V. lactea, and that substrate requirements, together with phylogenetic differences and the size of soredia, differentiate these species. The chemical composition of secondary lichen metabolites in both analyzed species showed similar variation and, therefore, this feature is not diagnostic in species recognition, although suggested by previous studies. The potential speciation of the two species seems to be caused by the shift in the substrate requirements.