Flavonoids are important secondary metabolites in fruits, and contribute to both fruit quality and human health. Characterization of genes involved in flavonoid synthesis is critical for genetic modification of the amount and composition of flavonoids in fruits. Here, we report on the isolation of genes encoding flavonoid 3'-hydroxylase (F3'H) in apple. Three genes, designated MdF3'H1, MdF3'H2-1, and MdF3'H2-2 were identified from cv. 'GoldRush' based on EST database assembly and BAC library screening. Each MdF3'H gene is composed of 3 exons and 2 introns and contains an open reading frame of 1,536 bp encoding a putative protein of 511 amino acids. MdF3'H1 shows 90% nucleotide sequence identities in coding region sequence with either MdF3'H2-1 or MdF3'H2-2. MdF3'H2-1 and MdF3'H2-2 share an identity of 99% nucleotide sequence identity in the coding region, and were identified to be alleles. An indel ∼540 bp in size was identified in the first intron of MdF3'H2-1 and MdF3'H2-2, and successfully used to develop gene-tag STS for MdF3'H2 gene. In addition, a gene-tag STS was also developed for MdF3'H1 based on a (CT)n repeat in the second intron.