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Photosynthetic carbon isotope discrimination is a non-destructive tool for investigating C₄ metabolism. Tuneable diode laser absorption spectroscopy provides new opportunities for making rapid, concurrent measurements of carbon isotope discrimination and CO₂ assimilation over a range of environmental conditions, and this has facilitated the use of carbon isotope discrimination as a probe of C₄ metabolism. In spite of the significant progress made in recent years, understanding how photosynthetic carbon isotope discrimination measured concurrently with gas exchange relates to carbon isotope composition of leaf and plant dry matter remains a challenge that requires resolution if this technique is to be successfully applied as a screening tool in crop breeding and phylogenetic research. In this review, we update our understanding of the factors and assumptions that underlie variations in photosynthetic …