. Mesophyll conductance (g m) has been shown to vary between genotypes of a number of species and with growth environments, including nitrogen availability, but understanding of g m variability in legumes is limited. We might expect g m in legumes to respond differently to limited nitrogen availability, due to their ability to fix atmospheric N2. Using online stable carbon isotope discrimination method, we quantified genetic variability in g m under ideal conditions, investigated g m response to N source (N2-fixation or inorganic N) and determined the effects of N source and water availability on the rapid response of g m to photosynthetic photon flux density (PPFD) and radiation wavelength in three genotypes of chickpea (Cicer arietinum). Genotypes varied 2-fold in g m under non-limiting environments. N-fed plants had higher g m than N2-fixing plants in one genotype, while g m in the other two genotypes was unaffected. g m response to PPFD was altered by N source in one of three genotypes, in which the g m response to PPFD was statistically significant in N-fed plants but not in N2-fixing plants. There was no clear effect of moderate water stress on the g m response to PPFD and radiation wavelength. Genotypes of a single legume species differ in the sensitivity of g m to both long- and short-term environmental conditions, precluding utility in crop breeding programmes.