The genus Panstrongylus includes 14 species widely distributed from Mexico to Argentina, some of them with great epidemiological significance as vectors of Chagas disease. We study the karyotype and the male meiotic process of Panstrongylus chinai, P. geniculatus, P. herreri, P. lignarius, P. megistus, P. rufotuberculatus and P. tupynambai. All species present the same sex mechanism (X1X2Y in males and X1X1X2X2 in females) and they also have 20 autosomes, with the exception of P. megistus that only presents 18 autosomes. The analysis of C-banding patterns and meiotic chromosome behaviour show a great level of variability allowing the identification of three clearly differentiated groups. In the first group, we only include P. megistus because of its unusual number of autosomes. The second group includes P. chinai, P. herreri, P. lignarius and P. rufotuberculatus. Their autosomes present terminal heterochromatic regions that appear scattered throughout the nucleus and associated with the sex chromosomes. Actually, P. herreri and P. lignarius can be considered cytogenetically identical. Our results are in agreement with morphological, ecological and molecular data indicating that they should be regarded as the same species. The third group only includes P. tupynambai that shows autosomes without C-positive regions. Panstrongylus geniculatus shares characters will all the three groups. Its karyotypic features are extremely polymorphic depending on their geographic origin. Some populations do not show any heterochromatic regions, while others exhibit few or several heterochromatic blocks. The chromosomal variability observed, together with its wide distribution and phenetic variability, suggest that P. geniculatus is a species complex comprising at least two distinct species. Considering the entire subfamily, the level of cytogenetic variation in Panstrongylus is lower than that observed in Triatoma but considerably more than that of Rhodnius, which is a very homogenous genus in terms of chromosome appearance and behaviour. This would endorse the closer relationship between Panstrongylus and Triatoma, and their divergence from Rhodnius, in accordance with current tribal classification. © 2002 Elsevier Science B.V. All rights reserved.