Photosynthesis is fundamental to all life on Earth. In terrestrial plants it relies exclusively on RuBisCO to fix atmospheric CO2 into the sugar-generating Calvin-Benson-Bassham cycle. RuBisCO however, binds with oxygen preferentially under favourable conditions which has led to the evolution of carbon concentrating mechanisms such as C4 photosynthesis. While being present in just 3% of global flora, the C4 cycle is responsible for around a quarter of primary productivity. This increased efficiency if engineered into C3 species such as rice would dramatically enhance yield. This objective is challenging as it involves introducing incompletely understood traits into C3 leaves including complex changes to their biochemistry, cell biology and anatomy. Quantitative genetics offers an under-explored route to identify regulators of these processes. Taking advantage of the natural variation present for C4 characteristics in the dicotyledon Gynandropsis gynandra, this thesis aims to carry out linkage mapping and heritability estimates of C4 traits. Additionally, this thesis describes the generation of a Multi-Parental Advanced Generation Inter-Cross population and tools for high-throughput phenotyping of vein density, a trait integral to the evolution of the C4 syndrome. Resources presented in this thesis can be used by the C4 community to compliment knowledge gained through functional genomics so that improvements can be made in our understanding of C4 photosynthesis.