Rapid and dramatic environmental change in transboundary ecosystems is creating opportunities for both cooperation and conflict not only between states but also among the many non-state actors – whether local, regional, or global – with ties to these areas. This is the case in the Mekong River Basin and Arctic circumpolar region, where environmental change is affecting the livelihoods of resource-dependent communities and complicating geopolitical relations between nation-states and regional actors. In both ecosystems, regional governance is underpinned by often unexamined spatial agendas and knowledge claims, raising questions about how these regions are imagined and understood, as well as about how different ways of knowing intersect with transboundary cooperation and legitimize or challenge dominant discourses associated with environmental geopolitics. Of particular interest are the opportunities for local and traditional ecological knowledges, construed broadly, to influence cooperative initiatives in these different contexts. A comparison of the Arctic and Mekong regions offers insights into how global environmental geopolitics are shifting, particularly in terms of movements away from a state-centric model of world politics to one that must account for diverse actors and interests operating at a range of spatial scales.