We combine evidence from small-scale experiments with a large-scale field survey to clarify the roles of biotic resistance and pre-adapted habitat niche segregation to the invasion success of the North American brook trout (Salvelinus fontinalis) in North European streams previously dominated by brown trout (Salmo trutta). Interspecific aggressions among the two species were negligible, yet there was distinct habitat niche segregation between them: brook trout occupied mainly pool habitats while brown trout tended to reside in fast-flowing riffles. Habitat niche segregation among brook trout and brown trout prevailed across a wide array of scales from experimental flumes to entire drainage systems, although the segregation pattern was weaker in the field. Habitat differentiation among the two species reflected their differential habitat requirements, suggesting that a match between a species’ niche requirements in its native range and habitat availability in the new environment is a prerequisite for understanding invasion success.