Since 1966 the triatomines were grouped in complexes and specific subcomplexes. Although the complex and subcomplexes not have taxonomic importance, should be monophyletic groups and cytogenetic tools have proved to be of great importance to characterize these species groupings. Based on this, this paper aims to describe the chromosomal characteristics and heterochromatic pattern of Matogrossensis and Rubrovaria subcomplexes, in order to contribute to the taxonomic and evolutionary relationships of these vectors. In this study, at least three males from each species (Triatoma baratai, Triatoma costalimai, Triatoma guazu, Triatoma jurbergi, Triatoma matogrossensis, Triatoma vandae, Triatoma williami, Triatoma carcavalloi, Triatoma circummaculata, Triatoma klugi, Triatoma pintodiasi and Triatoma rubrovaria) were analyzed by means analyzed by means of cytogenetic techniques of C-banding. All species showed the same cytogenetic characteristics: 22 chromosomes, low variation in the size of autosomes, sex chromosome Y larger than X, initial prophase composed of only one heterochromatic chromocenter formed by the sex chromosomes X and Y (except for T. pintodiasi that presented the sex chromosomes individualized during all stages of prophase) and presence of constitutive heterochromatin restricted to sex chromosome Y. These characteristics, although common to Matogrossensis and Rubrovaria subcomplexes allow to distinguish these species of species grouped in most of South America subcomplexes, as Brasiliensis, Maculata, Sordida and Insfestans. Thus, the cytogenetic analysis was of extreme importance to differentiate both subcomplexes of the other subcomplexes of South America. However, probably due to evolutionary proximity existing between these subcomplexes was not possible to observar species differences that make up the Matogrossensis subcomplex of the Rubrovaria subcomplex. Therefore, we emphasize that new comparative analyzes, as experimental hybrid crosses and molecular cytogenetic analysis are necessary to clarify the evolutionary relationship between these important subcomplexes of vectors.