The phylogenetic relationships among species of Triatoma matogrossensis subcomplex (T. baratai, T. guazu, T. matogrossensis, T. sordida, T vandae, and T. williami) was addressed by using fragments of cytochrome oxidase I (COI), 16S rDNA (16S), and cytochrome b (Cytb) through Bayesian and parsimony analyses. We did not recover a monophyletic T matogrossensis subcomplex, and their members were found clustered in three strongly supported clades, as follows: i) T. jurbergi + T. matogrossensis + T. vandae + T. garciabesi + T. sordida; ii) with T guasayana as the sister group of clade (i); and iii) T. wilhami + T. guazu, however not closely related to the clade formed by the previously mentioned species. The other two endemic species from Central-Western Brazil, T. baratai and T. costalimai, were not recovered with strong clade support as related to other members of this subcomplex. Results call for a further revision in the classification of the subcomplexes within the genus Triatoma.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Sao Paulo State Univ UNESP, Sch Pharmaceut Sci, Dept Biol Sci, Araraquara, SP, BrazilUniv Fed Rio de Janeiro, Dept Zool, Inst Biol, BR-21941 Rio De Janeiro, RJ, BrazilUniv Sao Paulo, Phys Inst Sao Carlos, Sao Carlos, SP, BrazilUniv Connecticut, Ctr Hlth, Dept Genet & Dev Biol, Farmington, CT USASao Paulo State Univ UNESP, Sch Pharmaceut Sci, Dept Biol Sci, Araraquara, SP, BrazilFAPESP: 10/50.355-1FAPESP: 10/17027-0FAPESP: 11/22378-0