Human activity often has profound effects on plant growth and evolution. Orchids are the most diverse group of flowering plants and are threatened by habitat fragmentation, over-harvesting, and urbanization. A population of Phaius flavus from Beikengding Mount (BM) in Fujian Province was divided into two patches by road construction, and its genetic characteristics were evaluated using restriction site-associated DNA sequencing (RAD-seq) data. The purpose of this study is to explore the impact of road construction on the evolution of different patches within a population. Analysis revealed that the genetic diversity of patch B was slightly higher than that of patch A in the BM population of P. flavus. Principal component and phylogenetic analyses, genetic structure, genetic differentiation, and bottleneck detection indicated phase-independent genetic differentiation between the two patches. Thus, the construction of the Y013 village road may have influenced different patches of this population on a genetic level. This study provides a rational basis for research on the impact of human activities on biodiversity conservation.