N₂ fixation by moss-associated cyanobacteria plays an important role in the nitrogen cycling of terrestrial ecosystems. Recent studies have mainly focused on boreal ecosystems; little is known about such association in other ecosystems. Moss-associated cyanobacteria are subject to rapid changes (hourly or less) in environmental conditions that may affect N₂fixation kinetics. Using a recently developed method (Acetylene Reduction Assays by Cavity ring-down laser Absorption Spectroscopy, ARACAS) with higher sensitivity and sampling frequency than the conventional method, we characterize short-term kinetics of N₂ fixation by cyanobacteria on moss carpets from warm and cold temperate forests. We report the identification of a heretofore unknown multispecies true-moss–cyanobacteria diazotrophic association. We demonstrate that short-term change in abiotic variables greatly influences N₂ fixation. We also show that difference in relative proportion of two epiphytic diazotrophs is consistent with divergent influences of temperature on their N₂ fixation kinetics. Further research is needed to determine whether this difference is consistent with a latitudinal trend.