Matthiola incana is a popular winter-flowering plant, and white is considered a valuable flower color for marketed cultivars. In this study, we aimed to identify the genes responsible for white flower coloration in six commercial cultivars of M. incana used as cut and potted flowers. The expression levels of chalcone synthase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase (ANS), and anthocyanidin 3-O-glucosyltransferase in the petals of ‘Kiss me White’ and ‘Pygmy White’ were 0%–48% lower than those in the purple flower ‘Vintage Lavender’, whereas the expression level of basic helix-loop-helix 2 (bHLH2) was two-fold higher. Significantly reduced expression levels of ANS were also detected in four other white flower cultivars: ‘Vintage White’, ‘Iron White’, ‘White Wonder No. 2’, and ‘Quartet White’. All investigated white flower cultivars had a single nucleotide deletion in the first exon of ANS, which we designated as ans-1. This generates a frameshift mutation and a nonsense codon. In addition to ans-1, ‘Kiss me White’ and ‘Pygmy White’ have a 481-bp insertion within bHLH2. This insertion has features of hAT-type transposable elements and was designated as dTmi1. All white flower cultivars contain the ans-1 mutation, whereas ‘Kiss me White’ and ‘Pygmy White’ are double mutants containing both bhlh2dTmi1 and ans-1. ‘Kiss me Yellow’, which accumulates carotenoids, but not anthocyanins, in its petals possesses the bhlh2dTmi1 allele, but not the ans-1 allele. Therefore, either bhlh2dTmi1 alone or ans-1 alone can lead to a deficiency in anthocyanin production in commercial cultivars of M. incana. We also developed co-dominant DNA markers that can distinguish between wild-type and mutant alleles of both bHLH2 and ANS. In combination with other previously developed markers that can distinguish between single- and double-flowered individuals, these markers will be useful for nursery plant management and breeding of commercial M. incana.