Road networks are increasing globally, and as such incur increased pressure on wildlife communities. Large‐scale road expansion and infrastructure development occurring throughout Asia (i.e. Belt and Road Initiative) will affect hundreds of threatened species, leading to an increase in wildlife‐vehicle collisions. However, only a limited number of studies have quantified road mortality within this threatened landscape. To rectify this, we monitored the spatial‐temporal patterns of vertebrate roadkill in the Dong Phayayen‐Khao Yai forest complex, an UNESCO World Heritage Site located in Thailand, considered a refuge for many globally threatened species. We ran 100 standardized road surveys between 2015 and 2017, on a 47.8‐km highway transect currently being expanded, and recorded a total of 1395 casualties from 124 species. After correcting for removal time and detection probability, we estimated an average of 9688 vertebrate mortalities per year. We observed a mortality peak during the dry season for almost all taxonomic groups, due to a combination of high bird and bat mortality events. All taxa showed significant spatial clustering, highlighting high‐risk road sections. We modelled the road and landscape features most likely to lead to higher mortality rates, disentangling small‐scale (250–500 m from road) from large‐scale effects (1000–2500 m). We recorded lower mortality rates and species diversity near forest sites in protected areas, representing either lower crossing events due to habitat quality or an already depleted environment. Spatial analyses provided information on hotspots and landscape factors, which can be used to inform future mitigations at multiple scales throughout the Southeast Asia region. Our study provides a baseline of the direct road impacts on vertebrate species in a tropical biodiversity hotspot, and we suggest that more studies be conducted to fully assess the direct road effects on the many threatened species in this landscape.