Environmental heterogeneity is a primary factor in determining soil microbial spatial patterns. However, few studies have demonstrated a link between environmental heterogeneity and the spatial patterns in free-living nitrogen-fixing bacteria, which are important in nitrogen cycling. Here, quantitative polymerase chain reaction and terminal restriction fragment length polymorphism were used to determine the spatial distribution of free-living nitrogen-fixing bacteria along a slope gradient. Plant communities were evaluated through field surveys, and basic soil properties were measured in the laboratory. Soil properties (e.g., soil available phosphorus, soil organic carbon, and total nitrogen) were higher in upper- and middle -slope soils than in lower-slope soils, while bacterial abundance was the opposite. Bacterial and plant communities all varied along the slope gradient. Redundancy analysis revealed that bacterial community composition was closely linked to pH, soil organic carbon, available phosphorus, and total nitrogen. These results indicate that free-living nitrogen-fixing bacterial communities show strong spatial structure along a slope gradient, and emphasize the importance of soil heterogeneity in affecting bacterial spatial patterns in a karst region.