Background and aims <p>Determining belowground species composition is vital in understanding interspecific competition or niche complementarity in grass-legume mixtures. This study explored the potential of Fourier Transform Infrared (FTIR) Spectroscopy to determine root composition in a three-species grass-legume mixture containing perennial ryegrass (<i>Lolium perenne</i> L<i>.</i>), white clover (<i>Trifolium repens</i> L.), and red clover (<i>Trifolium pratense</i> L<i>.</i>) under field conditions.</p> Methods <p>Artificial root mixtures (Artmix) (<i>n</i> = 66) and pure root (Pure) samples (<i>n</i> = 90) of three species were prepared using roots collected from a field experiment in Denmark. Two FTIR models were calibrated using Artmix and Pure samples, and validated with leave-one-out and blind validation (BV) methods. The Artmix FTIR model was used to determine the belowground species composition of mixed root biomass samples, and we related the belowground and aboveground species compositions.</p> Results <p>FTIR spectra (600 – 4000 cm⁻¹ wavenumbers) of three species were successfully differentiated in PCA and cluster analysis. Both Artmix and Pure FTIR models exhibited strong predictive performance in calibration (high R² [≥ 0.96], excellent RPD [&gt; 5], and low RMSEP [&lt; 5]). During BV, the Artmix model showed marginally better performance (RMSECV = 3–5; Bias = –1 to 2) than Pure model (RMSECV = 3–8; Bias = –6 to 2).</p> Conclusion <p>This study showed that FTIR spectroscopy is a promising approach to determine species-specific roots in grass-legume mixtures. It has the potential to give improved insight into species complementarity and thus could be used to design mixtures aiming at improving nutrient use efficiency, yield stability, and soil C sequestration potential.</p>