Rationale Dental calculus (mineralised dental plaque) is composed primarily of hydroxyapatite. We hypothesise that the carbonate component of dental calculus will reflect the isotopic composition of ingested simple carbohydrates. Therefore, dental calculus carbonates may be an indicator for sugar consumption, and an alternative to bone carbonate in isotopic palaeodiet studies. Methods We utilised Fourier transform infrared attenuated total reflectance analysis to characterise the composition and crystallisation of bone and dental calculus before isotope analysis of carbonate. Using a Sercon 20-22 mass spectrometer coupled with a Sercon GSL sample preparation system and an IsoPrime 100 dual inlet mass spectrometer plus Multiprep device to measure carbon, we tested the potential of dental calculus carbonate to identify C-4 resources in diet through analysis of delta C-13 values in paired bone, calculus and teeth mineral samples. Results The modern population shows higher delta C-13 values in all three tissue carbonates compared to both archaeological populations. Clear differences in dental calculus delta C-13 values are observed between the modern and archaeological individuals suggesting potential for utilising dental calculus in isotope palaeodiet studies. The offset between dental calculus and either bone or enamel carbonate delta C-13 values is large and consistent in direction, with no consistent offset between the delta C-13 values for the three tissues per individual. Conclusions Our results support dental calculus carbonate as a new biomaterial to identify C-4 sugar through isotope analysis. Greater carbon fractionation in the mouth is likely due to the complex formation of dental calculus as a mineralized biofilm, which results in consistently high delta C-13 values compared to bone and enamel.