Legumes are used in forage agriculture to replace mineral N fertilizer by biological N fixation. We hypothesised that N addition through biological N fixation may have different effects on soil organic matter (SOM)112 quantity and composition compared to mineral N fertilization. In this study we introduced lucerne into grasslands and aimed to examine the resulting effects on C stocks and the molecular C signatures of roots and soil. Lucerne, cocksfoot and tall fescue were grown for five years as monocultures or mixtures. Grass monocultures were N-fertilised (SNF), while lucerne monoculture and mixtures received N through biological N-2 fixation (BNF). For root and soil samples from 30 cm depth, we analysed quantity and composition of (1) non-cellulosic neutral carbohydrates after acid hydrolysis and (2) lignin after CuO oxidation. Our data showed species-specific chemical signatures for roots extracted from soil. Lucerne presence increased the N content of roots and lowered their lignin/N ratio. Contrasting root input over four growing seasons did not impact SOM stocks, as they were similar in all treatments. We observed different chemical signatures for soil under mixtures as compared to lucerne monoculture. In particular, the state of SOM degradation was enhanced under mixtures. A greater input of higher quality litter to soils receiving BNF did not increase SOM storage. When lucerne was grown in mixture with grass, we observed lignin and carbohydrate signatures that indicated a more advanced state of degradation as compared to monocultures. Therefore, we suggest that the introduction of lucerne into grassland influences OM degradation more than its stabilization. After four growing seasons, SOM molecular signatures were not significantly influenced by type of N fertilization and showed little difference among the treatments despite species specific root composition.