Intra- and inter-specific variation in CO2 assimilation rate (A) in Triticum spp. is well documented for reproductive growth stages. Research was conducted to characterize early vegetative photosynthetic variation in a diverse set of cultivated hexaploid wheat (T. aestivum L.) germplasm and in wild tetraploid (T. dicoccoides Korn) and hexaploid x tetraploid populations. Choice of hexaploid genotypes was based on maximum genetic distance between cultivars within the HRW and SRW wheat classes of the USA. The tetraploid material was produced by hybridizing two accessions of T. dicoccoides previously shown to differ widely in A and A/Chl but with similar leaf morphology. Genetic variability in the HRW and SRW gene pools was attributed to more recently developed descendent lines and unrelated lines rather than parental lines. Phenotypic distributions for A, stomatal conductance (gs), and internal CO2 concentration (Ci) in the F2 tetraploid population were continuous and showed transgressive segregation, reflecting quantitative inheritance with intermediate heritability. Variability in A was not associated with chlorophyll content or CO2 supply to the mesophyll measured as Ci. Genetic variability in A was also observed in the interspecific backcross population, 2*TAM W-101/PI 428109, thereby providing a germplasm pool to select for high A while restoring the D genome of hexaploid wheat. These results suggest that genetic improvement of vegetative assimilation rate is feasible in hexaploid wheat via homologous transfer from an alien source.