Traditional methods of root biomass field investigation are difficult to implement in karst forests, and root excavation methods cause high uncertainties, thereby resulting in a lack of data. In the present study, root biomass and spatial distribution patterns of woody plants in a karst evergreen and deciduous broadleaf and mixed forest in central Guizhou Province, southwestern China were analyzed by building root biomass allometric functions and using vegetation plot surveys. Root biomass regression models were established based on the root data of 106 trees of five dominant species (Machilus cavaleriei, Platycarya strobilacea, Carpinus pubescens., Itea yunnanensis, and Lithocarpus confinis.), 34 shrubs of three dominant species (Zanthoxylum dimorphophyllum, Stachyurus obovatus, and Rhamnus heterophylla), and 34 lianas of two dominant species (Dalbergia hancei Benth. and Rosa cymosa). The estimated root biomass of woody plants in the karst forest was 22.72 Mg/hm². Trees with 22.57 Mg/hm² root biomass accounted for 99.30% of the total forest root biomass and were the major root biomass contributors, with the five dominant tree species comprising 86.54% (19.67 Mg/hm²) of the total forest root biomass. The root development level of different species is a significant factor that influences spatial distribution patterns of root biomass. This study provides a new way to comprehensively estimate belowground vegetation biomass and carbon storage in karst regions.