Lichens, symbiotic organisms with a high tolerance to harsh environments, possess a greater diversity of sterols than other organisms. Sterols are involved in maintaining membrane integrity, hormone biosynthesis, and signal transduction. (1) Background: A characteristic feature of lichen sterols is a high degree of unsaturation, which influences membrane properties. Desaturases play an important role in the synthesis of unsaturated sterols, in particular, sterol C-5 desaturase (ERG3), which controls the conversion of episterol to ergosterol. Earlier, we demonstrated that the treatment of the lichen Peltigera canina with low and elevated temperatures results in changes in the levels of episterol and ergosterol. (2) Methods: Here, for the first time, we identified ERG3 in P. canina and, using an in silico analysis, we showed that PcERG3 belongs to the superfamily of fatty acid hydrolyases. A phylogenetic analysis was conducted to determine the evolutionary relationships of PcERG3. (3) Results: A phylogenetic analysis showed that PcERG3 clusters with ERG3 from other Peltigeralian and non-Peltigeralian lichens and also with ERG3 from free-living fungi. This suggests that PcERG3 has an ancient evolutionary origin and is related to fungi with lichenized ancestors, e.g., Penicillium. The differential expression of PcERG3 in response to temperature stress, a dehydration/rehydration cycle, and heavy metal exposure suggests that it plays a crucial role in maintaining the balance between more and less saturated sterols and, more generally, in membrane functioning. The multifaceted response of P. canina to abiotic stresses was documented by simultaneously measuring changes in the expression of PcERG3, as well as the genes encoding the heat shock proteins, PcHSP20 and PcHSP98, and PcSOD1, which encodes the antioxidant enzyme superoxide dismutase. (4) Conclusions: These findings suggest that PcERG3 is similar to the sterol C-5 desaturases from related and free-living fungi and plays important roles in the molecular mechanisms underlying the tolerance of lichens to environmental stress. Keywords: lichen; Peltigera canina; sterol biosynthesis; PcERG3; heat shock proteins; SOD1.