There is an increasing emphasis worldwide on breeding wheat cultivars with higher biomass and grain yield, whilst reducing excessive input of N fertilizers. Modern plant breeding has narrowed the genetic base of bread wheat, so exploring new sources of genetic variation for leaf photosynthesis traits and biomass to underpin grain yield improvement has become necessary. Our objectives were to quantify the genetic variability in a range of landrace-derived lines and elite bread wheat genotypes in flag-leaf photosynthesis traits, biomass and N-use efficiency for potential application in pre-breeding. Sixteen bread wheat genotypes including elite spring wheat cultivar Paragon × landrace crosses were tested in two field experiments in the UK. Averaging across years, pre-anthesis flag-leaf photosynthesis rate ranged amongst genotypes from 26.0 to 31.5 μmol/m2/s (P < 0.001) and post-anthesis from 14.2 to 18.6 μmol/m2/s (P < 0.001). Two landrace-derived lines had greater post-anthesis flag-leaf photosynthesis rate (P < 0.001) than their elite parent Paragon. There was a trend for a positive correlation among genotypes between post-anthesis flag-leaf photosynthesis rate and biomass (r = 0.47, P = 0.06). Two landrace derivatives showed higher post-anthesis flag-leaf stomatal conductance than Paragon. Our results indicated that introgression of traits from wider germplasm into elite UK modern wheat germplasm offers scope to raise flag-leaf photosynthetic rate and biomass.