Plantterpenoid synthase (TPS)family genes participate in metabolite synthesis, hormones, gossypol, etc. Here, we genome-widely identifiedTPSfamily genes in 12 land plant species. Four hundred and thirty TPS-related genes were divided into seven subfamilies. TheTPS-cin Bryophytes was suggested to be the earliest subfamily, followed by theTPS-e/fandTPS-hpresence in ferns.TPS-a, the largest number of genes, was derived from monocotyledonous and dicotyledonous plants. Collinearity analysis showed that 38 out of the 76TPSgenes inG. hirsutumwere collinear withinG. arboreumandG. raimondii. Twenty-oneGhTPS-agenes belong to the cadinene synthase (GhCDN)subfamily and were divided into five groups, A, B, C, D, and E. The specialcis-elements in the promoters of 12GhCDN-Agenes suggested that the JA and ethylene signaling pathways may be involved in their expression regulation. When 12GhCDN-Agenes were simultaneously silenced through virus-induced gene silencing, the glandular color ofGhCDN-A-silenced plants was lighter than that of the control, supported by a gossypol content decrease based on HPLC testing, suggesting thatGhCDN-Asubgroup genes participate in gossypol synthesis. According to RNA-seq analysis, gossypol synthesis-related genes and disease-resistant genes in the glandular variety exhibited upregulated expression compared to the glandless variety, whereas hormone signaling-related genes were downregulated. All in all, these results revealed plantTPSgene evolution rules and dissected theTPSsubfamily,GhCDN-A, function in gossypol synthesis in cotton.