Background: Commercially available poinsettia (Euphorbia pulcherrima) varieties prevalently accumulate cyanidin derivatives and show intense red coloration. Orange-red bract color is less common. We investigated four cultivars displaying four different red hues with respect to selected enzymes and genes of the anthocyanin pathway, putatively determining the color hue. Results: Red hues correlated with anthocyanin composition and concentration and showed common dark red coloration in cultivars 'Christmas Beauty' and 'Christmas Feeling' where cyanidin derivatives were prevalent. In contrast, orange-red bract color is based on the prevalent presence of pelargonidin derivatives that comprised 85% of the total anthocyanin content in cv. 'Premium Red' and 96% in cv. 'Harvest Orange' (synonym: 'Orange Spice'). cDNA clones of flavonoid 3'-hydroxylase (F3'H) and dihydroflavonol 4-reductase (DFR) were isolated from the four varieties, and functional activity and substrate specificity of the corresponding recombinant enzymes were studied. Kinetic studies demonstrated that poinsettia DFRs prefer dihydromyricetin and dihydroquercetin over dihydrokaempferol, and thus, favor the formation of cyanidin over pelargonidin. Whereas the F3'H cDNA clones of cultivars 'Christmas Beauty', 'Christmas Feeling', and 'Premium Red' encoded functionally active enzymes, the F3'H cDNA clone of cv. 'Harvest Orange' contained an insertion of 28 bases, which is partly a duplication of 20 bases found close to the insertion site. This causes a frameshift mutation with a premature stop codon after nucleotide 132 and, therefore, a non-functional enzyme. Heterozygosity of the F3'H was demonstrated in this cultivar, but only the mutated allele was expressed in the bracts. No correlation between F3'H-expression and the color hue could be observed in the four species. Conclusions: Rare orange-red poinsettia hues caused by pelargonidin based anthocyanins can be achieved by different mechanisms. F3'H is a critical step in the establishment of orange red poinsettia color. Although poinsettia DFR shows a low substrate specificity for dihydrokaempferol, sufficient precursor for pelargonidin formation is available in planta, in the absence of F3'H activity.