Abstract: Twenty-four unigene sequences shared high homology with bHLH transcription factor protein involved in plant anthocyanin biosynthesis were obtained from previous-constructed tree peony (Paeonia lutea) petal transcriptome database and named as PlbHLH1~24. By amino acid sequence comparing and phylogenetic tree analysis, it showed that PlbHLH3 was considered to be related to regulate anthocyanin biosynthesis, contained a 2 040 bp ORF encoding 679 amino acid residues with typical bHLH structural domain; the predicted protein sequence of PlbHLH3 also shared high similarity with other bHLH transcription factor that related to anthocyanin biosynthesis such as VvMYC1 and MdbHLH3. Relative Real-Time PCR analysis indicated that PlbHLH3 expressed in different tissues of P. lutea and P. delavayi, the expression in carpel, anther and petal was significantly higher than that in sepal and leaf; PlbHLH3 reached the highest abundance at early stage of P. lutea,. For P. delavayi, the PlbHLH3 reached the lowest level at early stage, and was significantly or extremely significantly lower than that in other four stages. In conclusion, it is inferred that PlbHLH3 might associate with the regulatory of anthocyanin biosynthesis in P. lutea and this would provide the basis for insight into the molecular mechanisms underlying tree peony yellow flower pigmentation.