In the present study, we characterized the phytochemical profile of six distinct ecotypes of Umbilicaria aprina Nyl. from Iran (include: Kivarestan, Mishan, Takht-e Nader, Tochal, Sabalan, and Sahand ecotypes) in order to ascertain whether the environmental differences can affect the chemical profile of this species. Based on the results of LC-ESI-MS/MS, 9 major chemical substances were identified: Orsellinic acid (1), Lecanoric acid (2), Evernic acid (3), Gyrophoric acid (4), Umbilicaric acid (5), Hiascic acid (6), Stictic acid (7), a member of Hiascic acid complex (8), and an unknown substance (9). The MS/MS fragmentation patterns of each substance were determined and proposed. Broad variability was observed in the chemical profiles of lichen samples according to the presence/absence of these substances. The results corroborate that the influence of ecological locality conditions for example altitude, slope aspect, light intensity, and temperature, on phytochemical variations among ecotypes of a particular lichen species like U. aprina are of great importance.