Abstract The genus Capsicum with its special flavour has been cultivated as a vegetable crop and spread throughout the American, African and Asian tropics. Because the genus Capsicum has various species and varieties, the clear species discrimination is always a focal point of research. In this study, analysis of phylogenetic relationship and evolution was been evaluated based on the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA). The ITS1-5.8S-ITS2 region was successfully amplified from 26 Capsicum annuum varieties and 4 C. eximium varieties using ITS universal primers. Results showed that the ITS1 region ranged from 244 bp to 251 bp, and (G + C) content (%) ranged from 38.89% to 58.33%; the 5.8S region ranged from 147 bp to 166 bp, and (G + C) content (%) ranged from 36.59% to 45.58%; and the ITS2 region ranged from 220 bp to 225 bp, and (G + C) content (%) ranged from 53.39% to 62.95%. According to the sequence alignment result, the highest dissimilarity rate among C. annuum varieties appeared between Coletti 2 and E41 EnZa, while among Capsicum eximium varieties, the highest dissimilarity rate appeared between Dangjo Gochu-1 and E49.9531-1. In the phylogenetic tree, C. annuum and C. eximium varieties formed one independent group by themselves, sharing only 79% identity with each other. Two subgroups were further separated in the C. annuum variety group. This result suggests that ITS region is efficient in the Capsicum phylogeny analysis. This work helps us further understand the phylogenetic relationship of Capsicum species.