Afforestation of degraded pastures can potentially enhance carbon sequestration, but little is known about the effects of eucalyptus plantations on soil organic matter (SOM) fractions. We used density and particle size SOM fractionations to evaluate changes in SOM for a chronosequence of eucalyptus plantations established on degraded pastures in two contrasting regions in southeastern Brazil. Declines in the content of soil C derived from the pasture (C4 photosynthetic pathway) and the accumulation of that derived from the eucalyptus (C3 photosynthetic pathway) were followed through 13C natural abundance of the SOM. The two study areas were in the Rio Doce River Valley, Minas Gerais State, namely: 1, Belo Oriente (BO, a region with lower elevation, higher mean annual temperature, lower forest productivities and dominated by clayey Typic Haplustoxs); 2, Virginópolis (VG, a region of higher elevation, higher forest productivities and dominated by clayey Rhodic Ustoxs). In the BO region the chronosequence included 0, 4.2, 13.2, 22.2, 32.0 and 34.2 years of eucalyptus cultivation and in the VG region soils were cultivated with eucalyptus for 0, 8.2, 19.2, 29.2 and 33.2 years. The accumulated cultivation time corresponds to about five rotations. In both regions the initial soil condition was represented by a site that was still under pasture. Soil samples collected at 0–10 and 10–20 cm depths were analyzed for: total organic carbon (TOC); C in the fulvic (FAF), humic (HAF) and humin (HF) fractions; C in the free (FLF) and occluded (OLF) light fractions; C associated with the heavy fractions, namely, the sand (SF), silt plus clay (S + CF) and clay (CF) fractions; and C in the microbial biomass (MB). Carbon stocks of virtually all SOM fractions were about twice as high in the VG region than in the BO region. Eucalyptus cultivation in the BO region increased C stocks in all SOM fractions, except for the MB fraction that was not altered and the SF fraction that declined with time of eucalyptus cultivation. Increases in TOC, FAF and HF organic C fractions were observed in the VG region. Mean annual accumulations across the entire cultivation period in the 0–10 cm soil layer showed virtually no difference averaging 0.22 Mg C ha −1 year −1 for the BO region and 0.23 Mg C ha −1 year −1 for the VG region. However, gains of TOC peaked by the end of the third rotation in the BO region and the second rotation in the VG region, so the TOC accumulation rate during the respective aggrading periods would correspond to 0.35 and 0.57 Mg C ha −1 year −1. There was no clear evidence that the more labile, fast cycling SOM fractions were more sensitive indicators of the impact of eucalyptus cultivation in the BO region, whereas in the VG region the FLF and OLF were more sensitive to the land use change than TOC.