Anthropogenic disturbances have long been acknowledged to be one of the primary threats to biodiversity worldwide, while little is still understood about how human-built infrastructure affects the gene flows and phylogeographic structure of plants. Such information is helpful for the conservation and restoration of human-disturbed ecosystems. Here we studied effects of a big river with a huge reservoir and two expressways on an island-endemic Primulina heterotricha (Gesneriaceae) on Hainan Island, China, one of the key parts of the glob ally important Indo-Burma biodiversity hotspot. By applying comparative phylogeography using one nuclear ribosomal DNA and two chloroplast DNA sequences, we estimated the levels of genetic diversity and differentiation in 176 and 117 individuals collected, respectively, before (in 2016) and after (in 2022) the construction of two expressways in Hainan Island, from the same eight populations of P. heterotricha. We found that Primulina heterotricha significantly increased nuclear genetic differentiation during the period 2016–2022, which coincides with the opening of the two expressways. Also notably, the sharing of ribotypes among the three groups of populations separated by the expressway network almost disappeared for the same period. Moreover, the changes in the significance of genetic barriers before and after road construction suggest that geographic isolation caused by both the reservoir and the expressways is key for understanding the present phylogeographical patterns of P. heterotricha. We provide direct evidence that anthropogenic infrastructures such as reservoirs and expressways have been capable of increasing genetic differentiation and, thus, modifying the phylogeographical pattern of P. heterotricha, in just a six-year period (or two generations of the study plant). To mitigate such negative pressure, we suggest establishing ecological corridors to enhance gene exchange between the two sides of the anthropogenic barriers.