AbstractAll grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C₃ or C₄ photosynthesis. As part of a multinational effort to introduce C₄ traits into rice to boost crop yield, candidate regulators of C₄ leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C₄-like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C₄ plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C₃ plants.