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China's Belt and Road Initiative (BRI) sets to create connections and build infrastructure across Eurasia, Asia, and parts of the African continent in its initial phase and is the largest infrastructure project of all time. Any infrastructure project on this scale will necessarily pass through ecofragile regions and key biodiversity areas (KBAs). This creates an imperative to identify possible areas of impact and probable effects on conservation values to facilitate adaptive planning and to mitigate, minimize, or avoid impacts. Using the highest resolution route maps of the BRI available, I overlaid the proposed road and rail routes on KBAs, protected areas, and predicted biodiversity hotspots for over 4138 animal and 7371 plant species. I also assessed the relationship between the proposed route with the distribution of mines across BRI countries and the proportion of deforestation and forest near routes. Infrastructure, especially mining, was clustered near the proposed route; thus, construction and development along the route may increase the size and number of mines. Up to 15% of KBAs were within 1 km of proposed railways. Thus, planned and probable development along the routes may pose a significant risk to biodiversity, especially because the majority of KBAs are unprotected. Many biodiversity hotspots for different taxa were near the route. These hotspots varied between taxa, making systematic management and environmental impact assessments an effective strategy for at least some taxa. A combination of planning and mitigation strategies will likely be necessary to protect the most important areas for biodiversity proximal to development, especially in currently unprotected KBAs and other regions that need protection. A fuller assessment of trade‐offs between conservation and other values will be necessary to make good decisions for each project and site being developed, including potentially modifying parts of the route to minimize impacts. Modification or foregoing of infrastructure may be needed if stakeholders consider the conservation costs too high. Article impact statement: Mitigating consequences to biodiversity of the largest ever global infrastructure project requires site‐based solutions and impact assessment. [ABSTRACT FROM AUTHOR]