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The Belt and Road Initiative (BRI) is the largest infrastructure development in human history. Given its scale of influence and infrastructure undertakings, it is set to bring far-reaching environmental impacts to regions such as Southeast Asia, one of the biologically richest and most diverse regions in the world. Knowing where and what biodiversity BRI will potentially affect is crucial to plan and address its negative impacts. Using BRI transport infrastructure spatial data, we conducted a GIS analysis of the potential BRI impacts in Southeast Asia on terrestrial and marine biodiversity indicators, including protected areas (PAs), Key Biodiversity Areas (KBAs), terrestrial ecoregions, forest cover, threatened species, and fragile ecosystems such as seagrasses, mangroves, and coral reefs. We assessed the potential impacts across four key distance thresholds (1, 5, 25, and 50 km "impact zones") on either side of the routes. For the terrestrial routes we assessed impacts for five different types of linear rail and road infrastructure development. Within 1 km of all routes 32 PAs, 40 KBAs and 29 ecoregions are intersected. While, 142 threatened species including 26 critically endangered species are within 5 km from new rail, which are also commonly found in frontier landscapes. In marine ecosystems 20 marine PAs and 16 KBAs are intersected by BRI marine routes. We conclude by discussing ways BRI could minimise its environmental impacts and utilise its political weight to advance conservation efforts in host nations. • BRI linear infrastructure impacts on biodiversity in Southeast Asia were mapped • Impacts were assessed within four impact zone distances for road, rail and shipping • 32 PAs, 40 KBAs and 29 ecoregions are within 1 km of terrestrial routes • 26 critically endangered and species are within 5 km of new rail • Vietnam and Peninsular Malaysia land and Indonesian marine routes are of concern [ABSTRACT FROM AUTHOR]