Most studies on bryophyte species in Syria were carried out for bryofloristic purposes; however, research on biochemical contents is still limited. The present study aimed to determine the fatty acids (FAs) content in four moss species (E. praelongum, R. murale, B. erythrorrhizon and A. riparium) widely spread in certain regions in Syria using GC-MS; fatty acids extraction by n-hexane was assisted by ultrasound to increase the extraction yield. The four moss species contained very long chain-polyunsaturtaed fatty acids (vl-PUFAs) larger than C18, like 20:5 (6–12%) and 20:4 (1–13%), and they also contained very long chain-saturated fatty acids (vl-SFAs), such as 24:0 (1–1.4%) and 26:0 (0–0.5%). FAs concentrations were highly different among the studied bryophytes. Palmitic acid 16:0 was identified as the main component in both E. praelongum and R. murale, while linoleic acid 18:2 and linolenic acid 18:3 constituted the main components in B. erythrorrhizon and A. riparium. FAs analysis showed a very high content of vl-PUFAs ranging between 12% and 24% for linoleic acid 18:2, from 19% to 21% for linolenic acid 18:3 and from 1% to 13% for arachidonic acid 20:4. Statistical analysis using Neighbor joining algorithm showed two clusters according to the original regions and confirmed the role of environmental factors on fatty acids content. The present study aimed to underline the importance of bryophytes as nutritionally rich species for further appropriate utilizations.