Habitat restoration resulting in changes in plant community composition or species dominance can affect the spatial pattern and variability of soil nutrients. Questions about how these changes in soil spatial heterogeneity develop over time at restoration sites, however, remain unaddressed. In this study, a geostatistical approach was used to quantify changes over time in the spatial heterogeneity of soil organic matter (SOM) across a 26-year chronosequence of tallgrass prairie restoration sites at FermiLab, outside of Chicago, Illinois. We used total soil N and C as an index of the quantity of SOM. We also examined changes in C:N ratio, which can influence the turnover of SOM. Specifically, the spatial structure of total N, total C, and C:N ratio in the top 10 cm of soil was quantified at a macroscale (minimum spacing of 1.5 m) and a microscale (minimum spacing of 0.2 m). The magnitude of spatial heterogeneity (MSH) was characterized as the proportion of total sample variation explained by spatially structured variation. At the macroscale, the MSH for total N decreased with time since restoration (r2= 0.99, p < 0.001). The decrease in spatial heterogeneity over time corresponded with a significant increase in the dominance of the C4 grasses. At the microscale, there was significant spatial structure for total N at the 4-year-old, 16-year-old, and 26-year-old sites, and significant spatial structure for total C at the 16-year-old and 26-year-old sites. These results suggest that an increase in dominance of C4 grasses across the chronosequence is homogenizing organic matter variability at the field scale while creating fine-scale patterns associated with the spacing of vegetation. Areas of higher soil moisture were associated with higher soil N and C at the two oldest restoration sites and at the native prairie site, potentially suggesting patches of increased belowground productivity in areas of higher soil moisture. This study is one of the first to report significant changes over time in the spatial structure of organic matter in response to successional changes initiated by restoration.