EMS-induced pepper green stem mutant gh1 and wild-type purple stem ‘Zhangshugang’ (ST-8) were used as materials in this study, and the anthocyanin content was determined. Transcriptome sequencing (RNA-seq) and real-time fluorescence quantitative PCR (qRT-PCR) were used to analyze the gene expression levels related to anthocyanin biosynthesis in pepper stems. The results showed that the anthocyanin content in the stem of mutant gh1 was significantly lower than that in the stem of ST-8. Compared with ST-8, gh1 obtained 1794 differentially expressed genes, including 1003 up-regulated genes and 791 down-regulated genes. Among them, genes related to anthocyanin biosynthesis pathway include 9 structural genes (DFR, UF3GT, F3H, ANS, CHI, CHS, C4H, PAL, 4CL) and 3 transcription factors (TT8, AN1, TTG1). Transcriptome expression analysis and qRT-PCR analysis were performed on differentially expressed genes related to anthocyanin synthesis. The results of the two analyses were basically the same. Four structural genes (CHS, CHI, DFR, UF3GT) and one transcription factor (AN1) were screened. It is speculated that these five genes might play an important role in the anthocyanin biosynthesis pathway in pepper stems.