Carbon isotope discrimination (Δ) during C3 photosynthesis is dominated by the fractionation occurring during CO2-fixation by the enzyme Rubisco. While knowing the fractionation by enzymes is pivotal to fully understanding plant carbon metabolism, little is known about variation in the discrimination factor of Rubisco (b) as it is difficult to measure using existingin vitromethodologies. Tuneable diode laser absorption spectroscopy has improved the ability to make rapid measurements of Δ concurrently with photosynthetic gas exchange. This study used this technique to estimateb in vivoin five tobacco (Nicotiana tabacumL. cv Petit Havana [N,N]) genotypes expressing alternative Rubisco isoforms. For transplastomic tobacco producingRhodospirillum rubrumRubiscobwas 23.8±0.7‰, while Rubisco containing the large subunit Leu-335-Val mutation had ab-value of 13.9±0.7‰. These values were significantly less than that for Rubisco from wild-type tobacco (b=29‰), a C3 species. Transplastomic tobacco producing chimeric Rubisco comprising tobacco Rubisco small subunits and the catalytic large subunits from either the C4 speciesFlaveria bidentisor the C3-C4 speciesFlaveria floridanahadb-values of 27.8±0.8 and 28.6±0.6‰, respectively. These values were not significantly different from tobacco Rubisco.