Karst areas are typical ecologically fragile areas, and stony desertification has become the most serious ecological and economic problems in these areas worldwide as well as a source of disasters and poverty. A reasonable sampling scale is of great importance for research on soil science in karst areas. In this paper, the spatial distribution of stony desertification characteristics and its influencing factors in karst areas are studied at different sampling scales using a grid sampling method based on geographic information system (GIS) technology and geo-statistics. The rock exposure obtained through sampling over a 150 m × 150 m grid in the Houzhai River Basin was utilized as the original data, and five grid scales (300 m × 300 m, 450 m × 450 m, 600 m × 600 m, 750 m × 750 m, and 900 m × 900 m) were used as the subsample sets. The results show that the rock exposure does not vary substantially from one sampling scale to another, while the average values of the five subsamples all fluctuate around the average value of the entire set. As the sampling scale increases, the maximum value and the average value of the rock exposure gradually decrease, and there is a gradual increase in the coefficient of variability. At the scale of 150 m × 150 m, the areas of minor stony desertification, medium stony desertification, and major stony desertification in the Houzhai River Basin are 7.81 km2, 4.50 km2, and 1.87 km2, respectively. The spatial variability of stony desertification at small scales is influenced by many factors, and the variability at medium scales is jointly influenced by gradient, rock content, and rock exposure. At large scales, the spatial variability of stony desertification is mainly influenced by soil thickness and rock content.