Pulses play a significant role in nitrogen cycling as they fix atmospheric N₂ through symbiosis. However, it is unknown whether there are differences in the ability of biological nitrogen fixation (BNF) among pulse species and individual cultivars. Here, we quantified the BNF ability of selected pulse cultivars and determined the effect on crop yield. A total of 25 species-cultivar combinations of chickpea (Cicer arietinum L.), dry bean (Phaseolus vulgaris L.), faba bean (Vicia faba L.), field pea (Pisum sativum L.), and lentil (Lens culinaris Medik.) were tested in 2008–2010. Pulses had a higher BNF in the wetter 2010 season, and a lower BNF in the drier 2009 season. In 2010, faba bean and chickpea had the highest BNF at 106 kg N ha^-1, followed by lentil, field pea, and dry bean at 87, 69, and 12 kg N ha^-1, respectively. Across years, field pea had the most stable BNF ability, fixing 55 kg N ha^-1 with an average seed yield of 2418 kg ha^-1. There are large differences in BNF and yield among cultivars within a species and the magnitude of the difference varied with years. Large genetic variability in BNF and yield suggest the possibility that pulse cultivars with a higher N₂-fixing ability and seed yield can be developed through selection of the N₂-fixing trait.