C4 photosynthesis overpasses C3 by a higher photosynthetic rate, along with a higher water and nitrogen use efficiency. The establishment of C4 photosynthesis requires modification of ancestral C3 in both anatomical and metabolic aspects, both are involved in many functional pathways which have not been fully demonstrated. This study aims to illustrate overall functional changes along the evolution of C4 photosynthesis, with a purpose to gain an overall view of what are required for C4 photosynthesis. To achieve this, this study assembled chromosome scale of genome sequencing of five Flaveria species, representing gradual evolutionary stages of C4 photosynthesis from C3 to C3-C4 and to C4. Here, result showed that the genome size gradually increases from C3 to intermediate to C4 species, which ranges from 0.5 G to 1.87 G. The enlarged genomes are mainly from transposable elements, with the number of protein coding gene are comparable along evolution. The copy of C4 version CA1 and PEPC1, as well as PEPC-k gained extra one, two and five copies in C4 species F. trinervia as a result of tandem duplication, which plays a positive role for the emergency of C4 photosynthesis. Besides, the evolutionary pattern of C4 gene on transcript abundance are consistent with that on protein abundance, suggesting transcriptional regulation is a key factor for establishing C4 photosynthesis.