Usnic acid contents in acetone extracts of 31 samples of lichen Parmelia flexilis collected from different altitudes were identified using thin layer chromatography (TLC) and determined by high performance liquid chromatography (HPLC). The usnic acid content varied in between highest 5.13% to lowest 1.66% in oven dried (80 °C) lichen samples. The species collected from lower altitudes all show high levels of usnic acid. The negative relationship between usnic acid and altitude was obtained. Statistically, it is revealed that there is a significant difference between average percentages of usnic acid in lichen samples with varying altitudes (p < 0.05). Beside these, the precipitation averages of the regions where the species have been collected were linked with the content of usnic acid. It is clear that lichens from the regions receiving the highest precipitation produced lower amounts of usnic acid. The results suggest that the production of secondary metabolite in lichens is altered due to the climatic variables like temperature and precipitation at different altitude gradients.