In the present study, we characterized the chemical variability of Arctoparmelia separata (Th. Fr.) Hale lichens are distributed in the Asian part of Russia and the northern part of North America to detect diversities within the species by high-performance liquid chromatography-mass spectrometry (HPLC-MS). A new chemotype of A. separata, containing stictic acid and related β-orcinol depsidones was identified. The difference in chemical composition correlated with geographical distribution, but not with morphological differences. The distinct geographical distribution between the A. separata chemotypes probably reflects their adaptation to environmental conditions. The possible functional role of stictic acid is briefly discussed. Keywords: Arctoparmelia separata; Secondary metabolites; HPLC-MS; Chemotypes; Asia; North America.