Over the past century, C3 woody plants and trees have increased in abundance in many semiarid ecosystems, displacing native C4 grasses. Livestock grazing, climatic fluctuations, and fire suppression are several reasons proposed for this shift. Soil carbon isotopic signatures are an ideal technique to evaluate carbon turnover rates in such ecosystems. On the gunnery ranges of Fort Huachuca in southeastern Arizona, study sites were established on homogeneous granitic alluvium to investigate the effects of fire frequency on δ¹³C values in surface soil organic matter (SOM). These ranges have had no livestock grazing for 50 years and a well-documented history of fires. Prosopis velutina Woot. (mesquite) trees have altered SOM δ¹³C pools by the concentration of plant nutrients and the addition of isotopically light litter. These soil carbon changes do not extend beyond canopy margins. Elevated total organic carbon (TOC), plant nutrient (N and P) concentrations, and depleted SOM δ¹³C values are associated with C3Prosopis on an unburned plot, which enables recognition of former Prosopis-occupied sites on plots with recent fire histories. Elevated nutrient concentrations associated with former Prosopis are retained in SOM for many decades. Surface SOM δ¹³C values indicate the estimated minimum turnover time of C4-derived carbon beneath large mature Prosopis is about 100–300 years. In contrast, complete turnover of original C3 carbon to C4 carbon under grasslands is estimated to take a minimum of 150–500 years. Our study confirms that C4 grass cover has declined over the past 100 years, although isolated C3 trees or shrubs were not uncommon on the historic C4-dominated grasslands. We find evidence in surface soil layers for a modern C3 plant expansion reflected in the substantial shift of SOM δ¹³C values from C4 grasses to C3 shrublands.