To investigate the molecular mechanism of cytoplasmic male sterile (CMS) in Capsicum annuum L., transcriptome Solexa sequencing was conducted on pepper flower buds of the CMS line 9704A and its correspondence homonuclear maintainer line 9704B. A gene that expressed 16 times higher in the maintainer liner 9704A than it is in the CMS line 9704B was identified. The cloned cDNA of this gene showed similarity to Chalcone Synthase Family genes with an overall sequence identity of 90% to Nicotiana sylvestris anther-specific gene-CHSLK. The expression of the gene in root, stem and leaf of the two peppers are tested by real-time fluorescence quantitative (RTFQ) RT–PCR. The result showed that the expression level of the gene in the stems of the two peppers was barely detectable, and in the root and leaf of the maintainer liner was very low, which confirmed that the gene is highly expressed specifically only in anther under normal condition. But the expression of this gene in roots and leaves of the CMS line 9704A is abnormally high, thus the abnormal CHS expression pattern in the CMS line may correlate to the male sterility. Key words: Capsicum annuum L.; chalcone synthase gene (CHS); quantitative analysis; male sterility