AbstractChemically defined media for yeast cultivation (CDMY) were developed to support fast growth, experimental reproducibility, and quantitative analysis of growth rates and biomass yields. In addition to mineral salts and a carbon substrate, popular CDMYs contain seven to nine B‐group vitamins, which are either enzyme cofactors or precursors for their synthesis. Despite the widespread use of CDMY in fundamental and applied yeast research, the relation of their design and composition to the actual vitamin requirements of yeasts has not been subjected to critical review since their first development in the 1940s. Vitamins are formally defined as essential organic molecules that cannot be synthesized by an organism. In yeast physiology, use of the term “vitamin” is primarily based on essentiality for humans, but the genome of the Saccharomyces cerevisiae reference strain S288C harbours most of the structural genes required for synthesis of the vitamins included in popular CDMY. Here, we review the biochemistry and genetics of the biosynthesis of these compounds by S. cerevisiae and, based on a comparative genomics analysis, assess the diversity within the Saccharomyces genus with respect to vitamin prototrophy.