The aim of this study was to determine the extent and geographical pattern of intraspecific chemical and genetic variability of the lichen Hypogymnia physodes by comparing populations from different habitats. We analyzed the secondary lichen substances and their relative concentrations using HPTLC and HPLC in samples collected from sites with different environmental conditions. We identified seven lichen substances: the cortical atranorin and chloroatranorin, and the medullary physodalic, physodic, protocetraric, 3-hydroxyphysodic, and 2′-0-methylphysodic acids. The samples were uniform qualitatively, which means that H. physodes has only one chemotype. We detected quantitative chemical differences between the samples without any geographical pattern. We investigated 21 samples in order to study the connection between genotypic diversity of populations and geographical distribution. We determined the sequences of five loci (ITS, nucSSU, nucLSU, mitSSU, EF1α). We found no significant genetic differentiation among populations collected from different areas.