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The red Chinese sand pear (<i>Pyrus pyrifolia</i> Nakai) is native to China and exhibits a unique fruit coloration pattern. Flavonoid 3′-hydroxylase (<i>F3′H</i>) catalyzes the hydroxylation of flavonoids, which subsequently determines the components of anthocyanins and the color of plant organs. Two genes encoding flavonoid 3′-hydroxylase (<i>F3′H</i>), <i>PpF3′HI</i> and <i>PpF3′HII</i>, have been identified in red Chinese sand pears. The coding regions for <i>PpF3′HI</i> and <i>PpF3′HII</i> were 1542 and 1536 bp in length, respectively. <i>PpF3′HI</i> shared 95% of its amino acid sequence identity with <i>PpF3′HII</i>, and a highly conserved P450 superfamily domain was found both in <i>PpF3′HI</i> and in <i>PpF3′HII</i>. Phylogenetic analysis showed that <i>PpF3′HI</i> and <i>PpF3′HII</i> clustered with MdF3′HI and MdF3′HII, respectively. <i>PpF3′H</i> genes were highly expressed in anthocyanin-enriched tissues such as young leaves, and transcription of <i>PpF3′H</i> genes corresponded to anthocyanin biosynthesis during the developmental stages, bagging treatment, and postharvest UV-B/visible irradiation treatment. A Y1H assay showed that PpMYB10 and PpHY5 could interact with the −419 bp to 0 bp and −746 bp to −396 bp fragments of the <i>PpF3′HI</i> promoter region, respectively. Understanding the mechanism of flavonoid hydroxylation patterns will, in turn, promote the development of new technologies for modifying flavonoid and anthocyanin composition in fruits.