[en] Replacing pastures (PA) with sugarcane (SG) has been deemed an agronomically feasible strategy for sugarcane expansion in Brazil. However, there are some uncertainties about the environmental impacts regarding this land use change (LUC), mainly related to soil organic matter (SOM), a key factor of environmental sustainability of Brazilian ethanol. LUC-related losses of SOM can overcome the C savings from biofuels. The molecular composition of SOM was evaluated to understand the C dynamics regarding LUC from PA to SG, using native vegetation (NV) as reference. Our study area was located in the south-central region of Brazil. Soil sampling was performed at three depths (0–0.1 m, 0.2–0.3 m and 0.9–1 m) in three representative sites with known LUC history and management practice since 1970. Pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS) was chosen to study SOM chemistry. Content and isotopic composition of soil organic C and N were also determined. The LUC caused decreases on C and N contents and on δ"1"3C isotopic values. Depth was the major factor that influenced SOM composition, while the influence of LUC was mainly evident in surface soils and diminished rapidly with depth. The main difference in SOM composition undergoing the conversion PA-SG was a higher contribution from compounds associated to fresh litter inputs. The high contribution from fresh litter, having a relatively low mean residence time and increasing decomposition rates, is probably a major factor that drives C losses in areas undergoing sugarcane expansion. - Graphical abstract: We chose 3 sites in Brazil (1) and collected soil samples in areas undergoing LUC pasture–sugarcane (2) to evaluate SOM composition using Py–GC/MS (3). The LUC causes shifts on SOM signature (4), highlighting the increase of fresh litter compounds in sugarcane areas (5). - Highlights: • Effect of land use change (LUC) on SOM composition in Brazilian soils • Py-GC/MS of NaOH extractable SOM under native vegetation, pasture and sugarcane • Regional differences were mainly evident at 1 m depth and related to BC • LUC mainly affected SOM composition in surface soils (0-0.1 m) • Conversion to sugarcane increased compounds associated to fresh litter on SOM