Methane is a potent greenhouse gas that is generated and consumed in anaerobic environments through the energy metabolism of methanogens and anaerobic methanotrophic archaea (ANME), respectively. Coenzyme F₄₃₀ is essential for methanogenesis, and a structural variant of F₄₃₀, 17²-methylthio-F₄₃₀ (F₄₃₀-2), is found in ANME and is presumably essential for the anaerobic oxidation of methane. Here we use liquid chromatography–high-resolution mass spectrometry to identify several new structural variants of F₄₃₀ in the cell extracts of selected methanogens and ANME. Methanocaldococcus jannaschii and Methanococcus maripaludis contain an F₄₃₀ variant (denoted F₄₃₀-3) that has an M⁺ of 1,009.2781. This mass increase of 103.9913 over that of F₄₃₀ corresponds to C₃H₄O₂S and is consistent with the addition of a 3-mercaptopropionate moiety bound as a thioether followed by a cyclization. The UV absorbance spectrum of F₄₃₀-3 was different from that of F₄₃₀ and instead matched that of an F₄₃₀ derivative where the 17³ keto moiety had been reduced. This is the first report of a modified F₄₃₀ in methanogens. In a search for F₄₃₀-2 and F₄₃₀-3 in other methanogens and ANME, we have identified a total of nine modified F₄₃₀ structures. One of these compounds may be an abiotic oxidative product of F₄₃₀, but the others represent naturally modified versions of F₄₃₀. This work indicates that F₄₃₀-related molecules have additional functions in nature and will inspire further research to determine the biochemical role(s) of these variants and the pathways involved in their biosynthesis.