Ecological connectivity in landscapes is crucial for plant diversity conservation. The barrier risk to ecological connectivity represents the risk to ecological connectivity loss or weakening, resulting from the barrier to biological information exchange among habitats. Therefore, clarifying the barrier risk to the ecological connectivity of plant diversity in space can reveal the spatial impacts of reduced ecological connectivity on plant diversity. This study analyzed effects of karst peak, river network, arable land, and impervious surface on plant diversity in karst natural, countryside, urban, and island landscapes in Guizhou Province with fragile environment. Then, we calculated the barrier distance of ecological connectivity to reveal the barrier risk to the ecological connectivity of plant diversity in space. The results showed that karst peak was the source of high plant diversity, and plant diversity could diffuse about 400 m around karst peaks. River network and arable land enhanced the connectivity among karst peaks to maintain plant diversity, and the effect on enhancing the connectivity was about 300 m and 450 m, respectively, while the weakening effect of impervious surface on connectivity was about 350 m. Based on the distance for plant diversity diffusing around karst peaks, the barrier distance of ecological connectivity was determined by the combination type of river network, arable land and impervious surface in landscapes. From low to high, the barrier risk to the ecological connectivity of plant diversity was about 1,110 m in the combination of river network and arable land, about 790 m in the combination of river network, arable land and impervious surface, about 520 min the combination of arable land and impervious surface, about 400 m in the combination of river network and impervious surface. Our findings clarify the barrier risk to the ecological connectivity of plant diversity in space, and provide a scientific basis for plant diversity conservation from the perspective of ecological connectivity.