De novosynthesis of thiamine (vitamin B1) in plants depends on the action of thiamine thiazole synthase, which synthesizes the thiazole ring, and is encoded by theTHI1gene. Here, we investigated the evolution and diversity ofTHI1in Poaceae, where C4 and C3 photosynthetic plants co-evolved. An ancestral duplication of THI1 is observed in Panicoideae that remains in many modern monocots, including sugarcane. In addition to the two sugarcane copies (ScTHI1-1andScTHI1-2), we identifiedScTHI1-2alleles showing differences in their sequence, indicating divergence betweenScTHI1-2aandScTHI1-2b. Such variations are observed only in the Saccharum complex, corroborating the phylogeny. At least fiveTHI1genomic environments were found in Poaceae, two in sugarcane,M. sinensis, andS. bicolor. TheTHI1promoter in Poaceae is highly conserved at 300 bp upstream of the start codon ATG and has cis-regulatory elements that putatively bind to transcription factors associated with development, growth, development and biological rhythms. An experiment set to compare gene expression levels in different tissues across the sugarcane R570 life cycle showed thatScTHI1-1was expressed mainly in leaves regardless of age. Furthermore,ScTHI1displayed relatively high expression levels in meristem and culm, which varied with the plant age. Finally, yeast complementation studies with THI4-defective strain demonstrate that onlyScTHI1-1andScTHI1-2bisoforms can partially restore thiamine auxotrophy, albeit at a low frequency. Taken together, the present work supports the existence of multiple origins of THI1 harboring genomic regions in Poaceae with predicted functional redundancy. In addition, it questions the contribution of the levels of the thiazole ring in C4 photosynthetic plant tissues or potentially the relevance of the THI1 protein activity.