Two telomere-to-telomere (T2T) gapless genomes of Capsicum annuum and its wild nonpungent relative Capsicum rhomboideum have been sequenced to investigate the evolution of fruit pungency and capsaicinoid biosynthesis in chili peppers. These genomes provide insights into the genetic basis of fruit pungency and the biosynthesis of capsaicinoids, which are alkaloids responsible for the heat in chili peppers. The T2T genomes reveal that Capsicum centromeres are extensively invaded by CRM retrotransposons, and that key biosynthesis genes in nonpungent species are disrupted. The genomes also show conserved placenta-specific accessible chromatin regions, which likely allow for tissue-specific biosynthetic gene regulation and capsaicinoid accumulation. The T2T genomic resources will accelerate chili pepper genetic improvement and help understand Capsicum genome evolution. The study also reveals that the capsaicinoid biosynthesis pathway in Capsicum evolved through tandem duplications of key genes and that the pathway is tissue-specific, occurring only in the placenta of the fruit. The T2T genomes provide a comprehensive view of the Capsicum genome, including centromeres, telomeres, and filled gaps, and highlight the unique features of Capsicum centromeres, such as their enrichment with Gypsy-LTR retrotransposons. The study also shows that the evolution of capsaicinoid biosynthesis in Capsicum is closely related to its divergence from other Solanaceae plants and that the pathway likely originated between 13.4 and 5 million years ago. The T2T genomes also reveal that the expression of capsaicinoid biosynthesis genes is tissue-specific, with high expression in the placenta of pungent peppers. The study provides a detailed analysis of the capsaicinoid biosynthesis pathway and its regulation, and highlights the importance of epigenetic and transcriptional mechanisms in tissue-specific gene expression. The T2T genomes are a significant milestone in plant genome research and will accelerate the study of chili peppers and their genetic improvement.Two telomere-to-telomere (T2T) gapless genomes of Capsicum annuum and its wild nonpungent relative Capsicum rhomboideum have been sequenced to investigate the evolution of fruit pungency and capsaicinoid biosynthesis in chili peppers. These genomes provide insights into the genetic basis of fruit pungency and the biosynthesis of capsaicinoids, which are alkaloids responsible for the heat in chili peppers. The T2T genomes reveal that Capsicum centromeres are extensively invaded by CRM retrotransposons, and that key biosynthesis genes in nonpungent species are disrupted. The genomes also show conserved placenta-specific accessible chromatin regions, which likely allow for tissue-specific biosynthetic gene regulation and capsaicinoid accumulation. The T2T genomic resources will accelerate chili pepper genetic improvement and help understand Capsicum genome evolution. The study also reveals that the capsaicinoid biosynthesis pathway in Capsicum evolved through tandem duplications of key genes and that the pathway is tissue-specific, occurring only in the placenta of the fruit. The T2T genomes provide a comprehensive view of the Capsicum genome, including centromeres, telomeres, and filled gaps, and highlight the unique features of Capsicum centromeres, such as their enrichment with Gypsy-LTR retrotransposons. The study also shows that the evolution of capsaicinoid biosynthesis in Capsicum is closely related to its divergence from other Solanaceae plants and that the pathway likely originated between 13.4 and 5 million years ago. The T2T genomes also reveal that the expression of capsaicinoid biosynthesis genes is tissue-specific, with high expression in the placenta of pungent peppers. The study provides a detailed analysis of the capsaicinoid biosynthesis pathway and its regulation, and highlights the importance of epigenetic and transcriptional mechanisms in tissue-specific gene expression. The T2T genomes are a significant milestone in plant genome research and will accelerate the study of chili peppers and their genetic improvement.