Inactivation of the gene (DFR-A) coding for dihydroflavonol 4-reductase (DFR) involved in the anthocyanin biosynthesis pathway results in a yellow bulb color in onion (Allium cepa L.). Three inactive alleles have previously been identified in onion, and in this study we identified three active and six inactive novel DFR-A alleles from extensive analyses of diverse onion germplasms. Of the germplasms analyzed, we identified a yellow mutant containing a 171-bp deletion in the promoter region, which we designated DFR-A PD . Critically reduced transcription of this mutant allele and perfect co-segregation with color phenotypes in segregating populations were observed. A second yellow mutant (DFR-A 5′DEL ) which we identified contained a 518-bp deletion covering exons 1 and 2, which played important roles in DFR function. Both 2- and 4-bp insertions in the coding region leading to the creation of pre-mature stop codons were also identified and designated DFR-A GT and DFR-A 2AT , respectively. A 1-bp substitution mutation (DFR-A K48N ) which changed a positively charged lysine residue into a neutral asparagine was identified. This lysine residue, a NADPH binding site, was strictly conserved in other species. In addition, insertion of a leucine residue around substrate binding sites and catalytic triad was identified in several yellow accessions and designated DFR-A TTA . Phylogenetic analysis of DFR-A alleles showed that all inactive alleles were independently derived from four different active alleles. In addition, the close relatedness and diversity of DFR-A mutants implied that all these mutations might have occurred after domestication of onions and had probably been maintained by artificial selection.