We analysed the chromosome complement and male meiosis in a natural population of Mepraia gajardoi (Hemiptera: Reduviidae: Triatominae). The normal karyotype of this species is 20 autosomes plus X1X2Y in males and X1X1X2X2 in females. We confirmed that M. gajardoi is cytogenetically strikingly different from M. spinolai (with which it was previously included), which supports the taxonomic separation of these two species. For the first time in the holocentric chromosomes of the subfamily Triatominae, a structural rearrangement was detected. Observations made during meiosis and mitosis indicated that one of eleven individuals had undergone a fusion between two non-homologous chromosomes, probably before germinal cell development. This autosomal translocation resulted in chromosomal irregularities such as an autosomal trivalent, autosomal univalents and chromosomal fragments, which altered the normal segregation of both autosomes and sex chromosomes. The chromosomal fragments resemble supernumerary or B chromosomes, which supports the idea that these chromosomes are remnants of structural rearrangements. The genetic consequences of translocations in holokinetic chromosomes and their role in the evolution of chromosomes in triatomines are also discussed.