Thiamin pyrophosphate is an essential cofactor of carbohydrate and branched-chain amino acid metabolism. Although its mechanistic role is well studied, the biosynthesis of thiamin has only recently been understood. Thiamin biosynthesis in Escherichia coli and Bacillus subtilis show some similarities, but diverge at key steps of thiazole formation. The biosynthesis of thiamin in eukaryotes is at a very early stage of understanding. Structural and mechanistic studies on thiamin biosynthetic enzymes have played a key role in increasing our understanding of thiamin pyrophosphate biosynthesis and have revealed unexpected evolutionary ties.