The lipid composition of two species of vascular plants, Equisetum variegatum Schleich. ex. Web. and E. scirpoides Michx., growing in the permafrost zone (Northeastern Yakutia, the Pole of Cold of the Northern Hemisphere), with average daily air temperatures in summer of +17.8 °C, in autumn of +0.6 °C, and in winter of −46.7 °C, was comparatively studied. The most significant seasonal trend of lipid composition was an accumulation of PA in both horsetail species in the autumn–winter period. Cold acclimation in autumn was accompanied by a decrease in the proportion of bilayer-forming lipids (phosphatidylcholine in the non-photosynthetic membranes and MGDG in photosynthetic membranes), an increase in the desaturation degree due to the accumulation of triene fatty acids (E. scirpoides), and an accumulation of betaine lipids O-(1,2-diacylglycero)-N,N,N-trimethylhomoserine (DGTS). The inverse changes in some parameters were registered in the winter period, including an increase in the proportion of “bilayer” lipids and decrease in the unsaturation degree. According to the data obtained, it can be concluded that high levels of accumulation of membrane lipids and polyunsaturated FAs (PUFAs), as well as the presence of Δ5 FAs in lipids, are apparently features of cold hardening of perennial herbaceous plants in the cryolithozone.