We know little about how shifts in tree species distribution and increases in forest fire intensity could affect the formation of pyrogenic organic matter (PyOM) or charcoal, one of the most important and persistent soil organic matter pools. This limitation arises partly because the role of the precursor wood in controlling PyOM formation is unclear. The current study shows how tree species and pyrolysis temperature (200, 300, 450 and 600 °C) interact to control the physicochemical structure of the PyOM experimentally derived from 13C/15N-enriched Pinus banksania and Acer rubrum, two important co-occurring gymnosperm and angiosperm tree species from North American boreal-temperate ecotones. Complementary physicochemical and thermodynamic measurements revealed different susceptibilities of the two wood species to charring, with PyOM intermediates formed at lower temperature from the pine, indicating that the tree species regulated the efficacy of PyOM formation. Particularly, we report high-resolution data describing the comprehensive chemical architecture of PyOM (both –C and –N) as they are formed, which are complemented by unique molecular-level insights on their labile fractions. We posit that the tree species and pyrolysis temperature interaction reflects distinctive anatomical features of the two major tree taxa, including greater effective porosity in gymnosperms that promote the loss of volatiles and enhance the heat exposure of bio-components. This study points to a higher temperature threshold for PyOM production in maple forests compared with pine forests, resulting in potentially more degradable and less sorbtive PyOM in ecotones dominated by the former species.