Guizhou province, famous for its karst landforms and rich biodiversity, is facing exceptional threats by intensive human activities, like many areas in China. As a result, Guizhou is a critical and priority area for biodiversity conservation. In this research, herbarium collections were used for evaluating the present protection effectiveness of Nature Reserves. 103,783 herbarium specimen records, belong to 7505 native vascular plant species were used as different layers in ArcGIS software at 0.15 × 0.15-degree grid resolution. In addition, 129 natural reserves were used in the aim of exploring biodiversity patterns, identifying species richness centers, endemism centers, and for biodiversity gap analysis. As a result, nine distinct critical biodiversity areas were recognized based on high species richness, high endemic and endangered species: eight patches with highest species number (>1000 species), four patches with high areas of endemism species (>15 species), and three small areas with high percentage of endangered species (>50 species). Results showed that some species richness centers and endemism centers are protected by the current Nature Reserve network in China. However, comparing the nine distinct biodiversity critical areas and current Nature Reserves, many gaps exist, and some places with high endemism or endangered species still need further attention. More Nature Reserves should be established to protect the gap areas. This research provided an overview of approaches to establishing biodiversity conservation priorities, and of strategies for filling gaps in the existing reserve network, and also can shed light on conservation tasks for other similar karst landform regions.