The ancient diffusion of cotton (Gossypium sp.) across the Old World is one of the most outstanding examples of social, environmental, technical and economic entanglement. The various trajectories of cotton products, including raw and processed seeds and fibres, are relevant markers of the circulation of knowledge, goods and people. However, understanding cotton diffusion in the past is limited by the fact that cotton products could have been produced locally and/or imported from different regions. Furthermore, cotton seeds and to a lesser extent, cotton fibres, are generally only found in charred form in archaeological contexts and this can be problematic as in some cases, the charring of plant remains results in an offset of the biogenic isotope values. In this study, the isotope composition of modern uncharred and experimentally charred cotton seeds that were grown in irrigated fields in Seville, Spain and greenhouses in Montpellier, France, was measured to establish the range of isotope fractionation that takes places across several parameters (temperature range: 50, 100, 150, 200, 225, 250, 275, 300, 325 and 350°C; time range: 2, 4, 8 and 16 hours). The results provide information on the extent to which carbonization effects measured δ13C and δ15N values and if such values can be used successfully to reconstruct the nature of the local growing environment.