Under future climate change, temperature will likely remain the most important factor driving boreal forest productivity and species distribution, with potential effects on the composition and structure of natural populations. However, thermal acclimation of physiological processes, particularly photosynthesis, could help mitigate the predicted decrease in net productivity associated with global warming (Way and Yamori, 2014; Girardin et al., 2016). Consequently, a better understanding of acclimation of both photosynthesis (An) and dark respiration (Rd) to warming might provide valuable information to predict the capacity of species to adapt under climate change, and help improve the predictive accuracy of process-based models (Slot et al., 2014; Lombardozzi et al., 2015). Investigations of thermal acclimation of An and Rd in natural conditions are limited compared with those conducted under controlled conditions. Studies conducted along a natural latitudinal gradient should be valuable to investigate growth habit and the thermal acclimation capacity at the intraspecific level, as it takes into account the seasonal fluctuations in temperature (monthly and daily temperature range) as well as the complex interactions with other climatic and non-climatic factors. The present study provides an exhaustive assessment of thermal acclimation of photosynthesis and dark respiration for a conifer under natural conditions, with valuable insights into the temperature responses of photosynthetic capacity attributes (Vcmax and Jmax) and mesophyll conductance under natural conditions. The present results are original in clearly showing a lack of thermal acclimation of net photosynthesis (An) and evidence for type II acclimation of dark respiration (Rd) in response to long-term (2–4 years) variation in temperature along the regional climatic gradient of 5.5 °C for both the southern and northern white spruce seed sources tested.