Stable isotope ratios in organisms can be used to estimate source contributions to the organism. However, during lipid synthesis light isotopes of carbon (12C) and hydrogen (1H) are preferentially incorporated into the lipids, potentially causing source contributions to be poorly estimated. Contrary to expectations and other published examples in animals, larval lampreys, which are basal vertebrates, have lipids which are enriched in heavy isotopes of carbon (13C), but still depleted in heavy hydrogen (deuterium; 2H). Four lamprey species were collected and their isotopes ratios of δ2H, δ13C, δ15N were measured in their muscle before and after lipid extraction. Larval lamprey of one species was collected every three months for a year from two streams in Maryland and the isotope ratios of muscle before and after lipid extraction, as well as the extracted lipid were measured. Muscle δ13C was positively related to C:N ratios in samples when lipids were not removed and δ2H was negatively associated with the percent hydrogen in a sample. As expected, the measured difference between muscle and lipid δ2H (ΔMLδ2H) was the same for all months and was 111‰ (SE = ± 21, n = 35), but the ΔMLδ13C was different between months (ANOVA, F3,53 = 5.05, p < 0.005) and was always negative. Our work suggests that while lipids are often enriched in 12C relative to muscle, this is not a universal rule. The physiological mechanism(s) for generating heavy carbon-backbones in lipids remains unknown and requires exploration.