From naturally infected pepper fruits collected in the field, the pathogen X responsible for pepper anthracnose was isolated and cultured. This pathogen was used to assess anthracnose resistancefor 112 commercial pepper varieties through in vitro fruit and seedling assays, with the goal of identifying resistant varieties. Transcriptome analysis was then conducted on the resistant varieties to further investigate their response to the pathogen. The morphological and molecular biological identification confirmed that the pathogen X was Colletotrichum fructicola. Among the 112 pepper varieties screened, three resistant materials, namely RX12-160, X-738-1, and RX22-1016, were identified, while the remaining 109 varieties were found to be susceptible materials. Further evaluation of the three resistant varieties at the seeding stage revealed that RX22-1016 exhibited strong resistance to anthracnose. Then, a serial of analysis, including transcriptome analysis, GO and KEGG pathway enrichment analyses, were performed on the RX22-1016. The processes with higher GO enrichment levels included photosynthetic electron transport in photosystem I, assembly of the NAD(P)H dehydrogenase complex, and photosynthesis, as well as light harvesting in photosystem I. In the KEGG enrichment analysis, pathways with higher enrichment levels included phenylpropanoid biosynthesis, glutathione metabolism, starch and sucrose metabolism, carbon fixation in photosynthetic organisms, and glycolysis/gluconeogenesis. The commonly differentially expressed genes were mainly concentrated in pathways such as phenylpropanoid biosynthesis and glutathione metabolism. Among these, the genes Caz03g16640, Caz01g15210, Caz04g07320, and Caz04g01720 showed upregulated expression during the early stages of interaction between RX22-1016 and the anthracnose pathogen. These genes were implicated in the synthesis of disease-resistance-related compounds, suggesting their potential role in the plant’s defense response.