The molecular and isotopic compositions of lipid biomarkers from cultured filamentous cyanobacteria (Phormidium, also known as Leptolyngbya) have been used to investigate the community and trophic structure of photosynthetic mats from alkaline hot springs of the Lower Geyser Basin at Yellowstone National Park. We studied a shallow-water coniform mat from Octopus Spring (OS) and a submerged, tufted mat from Fountain Paint Pots (FPP) and found that 2-methylhopanepolyols and mid-chain branched methylalkanes were diagnostic for cyanobacteria, whereas abundant wax esters were representative of the green non-sulphur bacterial population. The biomarker composition of cultured Phormidium-isolates varied, but was generally representative of the bulk mat composition. The carbon isotopic fractionation for biomass relative to dissolved inorganic carbon (DIC; ɛCO2) for cultures grown with 1% CO2 ranged from 21.4 to 26.1 and was attenuated by diffusion limitation associated with filament aggregation (i.e. cell clumping). Isotopic differences between biomass and lipid biomarkers, and between lipid classes, depended on the cyanobacterial strain, but was positively correlated with overall fractionation. Acetogenic lipids (alkanes and fatty acids) were generally more depleted than isoprenoids (phytol and hopanoids). The δ13CTOC for OS and FPP mats were somewhat heavier than for cultures (−16.9 and −23.6, respectively), which presumably reflects the lower availability of DIC in the natural environment. The isotopic dispersions among cyanobacterial biomarkers, biomass and DIC reflected those established for culture experiments. The 7-methyl- and 7,11-dimethylheptadecanes were from 9 to 11 depleted relative to the bulk organic carbon, whereas 2-methylhopanols derived from the oxidation-reduction of bacteriohopanepolyol were enriched relative to branched alkanes by approximately 5–7. These isotopic relationships survived with depth and indicated that the relatively heavy isotopic composition of the OS mat resulted from diffusion limitation. This study supports the suggestion that culture studies can establish valid isotopic relationships for interpretation of trophic structure in modern and ancient microbial ecosystems.