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Pansy (Viola × wittrockiana Gams.) is an important and attractive ornamental plant with a wide variety of flower colors. To date, the molecular genetic understanding of its colorful petal pigment patterns remains largely unknown. In this study, we analyzed the bicolor petals of "Mengdie" in cytological, physiological, and transcriptomic aspects. Results showed that the difference of flower colors was mainly determined by the pigment distribution in the epidermal cells. Pigment analysis indicated that anthocyanins had strong correlations with color parameters, which acted as the main factor in flower coloration. Comparative transcriptome analysis found a total of 43,908 unigenes with the mean length of 682 bp. There were 24,323, 16,668, 8507, and 7680 unigenes annotated in the Nr, Swiss-Prot, KOG, and KEGG databases, respectively. Differential expression genes (DEGs) showed that the expression of anthocyanin late biosynthesis genes (LBGs), VwF3′H, VwF3′5′H, and VwUFGT, would be likely to play a major role in the color formation of pansy. The expression patterns of selected DEGs were verified by qRT-PCR. This study contributes an excellent insight into molecular mechanism of pigment biosynthesis and provides some useful information for flower color modification in pansy. [ABSTRACT FROM AUTHOR]