Plant species may show a preference for uptake of particular nitrogen (N) forms, but little is known about how N form preference is influenced by soil temperature. Potential future changes in soil N form availability and plant N form preference in warmer soils might shift competitive interactions among forest tree species. We compared the N uptake and growth of three conifer species from contrasting environments grown at rhizosphere temperatures of 10, 16 or 20 °C and supplied with ammonium (NH4+) or nitrate (NO3−) or a mix of arginine and alanine. Short-term N uptake was assessed using ion-selective microelectrodes and application of 15N, and long-term uptake was assessed by plant N status. Species exhibited preferences for particular N forms, and these preferences related to the N form most available in native soils. Specifically, Douglas-fir (Pseudotsuga menziesii(Mirb.) Franco) showed a preference for nitrate (a N form commonly found in warmer areas), Sitka spruce (Picea sitchensis(Bong.) Carr.) preferred ammonium (a N form abundant in cold soils) and Engelmann spruce (Picea engelmanniiParry ex Engelm.) showed a preference for ammonium and organic N (organic N is often abundant in cold soils). Relative N form preference, as indicated by plant growth, changed with temperature in some species, indicating that these species could acclimate to changing rhizosphere temperatures. Understanding how conifers utilize available soil nutrients at different temperatures can help to predict species' future performance as soil temperatures rise.