RNA-seq, the measurement of steady-state RNA levels by next generation sequencing, has enabled quantitative transcriptome analyses of complex traits in many species without requiring the parallel sequencing of their genomes. The complex trait of C-4 photosynthesis, which increases photosynthetic efficiency via a biochemical pump that concentrates CO2 around RubisCO, has evolved convergently multiple times. Due to these interesting properties, C-4 photosynthesis has been analyzed in a series of comparative RNA-seq projects. These projects compared both species with and without the C-4 trait and different tissues or organs within a C-4 plant. The RNA-seq studies were evaluated by comparing to earlier single gene studies. The studies confirmed the marked changes expected for C-4 signature genes, but also revealed numerous new players in C-4 metabolism showing that the C-4 cycle is more complex than previously thought, and suggesting modes of integration into the underlying C-3 metabolism.