C₄ 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 C₃ to C₄ photosynthesis. Chromosome-scale genome sequence analyses reveal a gradual increase in genome size during the evolution of C₄ photosynthesis attributed to the expansion of transposable elements. Systematic annotation of genes encoding C₄ enzymes and transporters identify additional copies of three C₄ enzyme genes through retrotranspositions in C₄ species. C₄ genes exhibit elevated mRNA and protein abundances, reduced protein-to-RNA ratios, and comparable translation efficiencies in C₄ species, highlighting a critical role of transcriptional regulation in C₄ evolution. Furthermore, we observe an increased abundance of ethylene response factor (ERF) transcription factors and cognate cis-regulatory elements associated with C₄ genes regulation. Altogether, our study provides valuable genomic resources for the Flaveria genus and sheds lights on evolutionary and regulatory mechanisms underlying C₄ photosynthesis.