Selective pressure exerted by a massive decline in atmospheric CO2 levels 55 to 40 million years ago promoted the evolution of a novel, highly efficient mode of photosynthetic carbon assimilation known as C-4 photosynthesis. C-4 species have concurrently evolved multiple times in a broad range of plant families, and this multiple and parallel evolution of the complex C-4 trait indicates a common underlying evolutionary mechanism that might be elucidated by comparative analyses of related C-3 and C-4 species. Here, we use mRNA-Seq analysis of five species within the genus Flaveria, ranging from C-3 to C-3-C-4 intermediate to C-4 species, to quantify the differences in the transcriptomes of closely related plant species with varying degrees of C-4-associated characteristics. Single gene analysis defines the C-4 cycle enzymes and transporters more precisely and provides new candidates for yet unknown functions as well as identifies C-4 associated pathways. Molecular evidence for a photorespiratory CO2 pump prior to the establishment of the C-4 cycle-based CO2 pump is provided. Cluster analysis defines the upper limit of C-4-related gene expression changes in mature leaves of Flaveria as 3582 alterations.