Abstract C 4 photosynthesis exemplifies convergent evolution of complex traits. Herein, we construct chromosome-scale genome assemblies and perform multi-omics analysis for five Flaveria species, which represent evolutionary stages from C 3 to C 4 photosynthesis. Chromosome-scale genome sequence analyses reveal a gradual increase in genome size during the evolution of C 4 photosynthesis attributed to the expansion of transposable elements. Systematic annotation of genes encoding C 4 enzymes and transporters identify additional copies of three C 4 enzyme genes through retrotranspositions in C 4 species. C 4 genes exhibit elevated mRNA and protein abundances, reduced protein-to-RNA ratios, and comparable translation efficiencies in C 4 species, highlighting a critical role of transcriptional regulation in C 4 evolution. Furthermore, we observe an increased abundance of ethylene response factor (ERF) transcription factors and cognate cis -regulatory elements associated with C 4 genes regulation. Altogether, our study provides valuable genomic resources for the Flaveria genus and sheds lights on evolutionary and regulatory mechanisms underlying C 4 photosynthesis.