Sedimentary 2-methyhopanes have been used as biomarker proxies for cyanobacteria, the only known bacterial clade capable of oxygenic photosynthesis and the only group of organisms found thus far to produce abundant 2-methylbacteriohopanepolyols (2-MeBHPs). Here, we report the identification of significant quantities of 2-MeBHP in two strains of the anoxygenic phototroph Rhodopseudomonas palustris. Biosynthesis of 2-MeBHP can occur in the absence of O(2), deriving the C-2 methyl group from methionine. The relative abundance of 2-MeBHP varies considerably with culture conditions, ranging from 13.3% of total bacteriohopanepolyol (BHP) to trace levels of methylation. Analysis of intact BHPs reveals the presence of methylated bacteriohopane-32,33,34,35-tetrol but no detectable methylation of 35-aminobacteriohopane-32,33,34-triol. Our results demonstrate that an anoxygenic photoautotroph is capable of generating 2-MeBHPs and show that the potential origins of sedimentary 2-methylhopanoids are more diverse than previously thought.