The goal of this study was to examine carbon isotope fractionations associated with lipid biomarkers of different iron-reducing bacteria. Experiments were conducted using a Geobacter metallireducens strain GS-15 (grown on acetate) and a Shewanella algae strain BrY (grown on lactate). Both organisms were grown anaerobically using ferric citrate as the electron acceptor. The δ13C of total biomass of GS-15 was −34.3‰, which was about 7.2‰ lower than the substrate acetate (δ13C=−27.1‰). The δ13C of total biomass of BrY was −30.0‰, which was about 7.1‰ lower than the substrate lactate (δ13C=−22.9‰). Isotopic fractionations (ε) between fatty acid sand biomass within the same strain were consistently greater for BrY (−10.9‰ to −15.5‰) than for GS-15 (−4.5‰ to −8.6‰). The lipid biomarker 10Me16:0, which was only found in GS-15 cultures, had an ε value of −5.39±0.73‰ (n=2). The differences in fractionation may reflect the carbon assimilation pathways by which the fatty acids were synthesized: the smaller fractionations in GS-15 may be associated with the tricarboxylic cycle, whereas the greater fractionations in BrY may be associated with the serine pathway. The difference in isotope fractionations between lipid biomarkers and total biomass of different iron-reducing bacteria may be used to enhance our understanding of the microbial community structure in an iron-reducing environment.