Spatial prioritizations are essential tools for conserving biodiversity in the face of accelerating climate change. Uncertainty about species' responses to changing climates can complicate prioritization efforts, however, and delay conservation investment. In an effort to facilitate decision-making, we identified three hypotheses about species' potential responses to climate change based on distinct biological assumptions related to niche flexibility and colonization ability. Using 314 species of North American birds as a test case, we tuned separate spatial prioritizations to each hypothesis and assessed the degree to which assumptions about biological responses affected the perceived conservation value of the landscape and prospects for individual taxa. We also developed a bet-hedging prioritization to minimize the chance that incorrect assumptions would lead to valuable landscapes and species being overlooked in multispecies prioritizations. Collectively, these analyses help to quantify the sensitivity of spatial prioritizations to different assumptions about species' responses to climate change and provide a framework for enabling efficient conservation investment despite substantial biological uncertainty.