The present study aimed to cytogenetically analyse five Ctenidae species Ctenus
ornatus (Keyserling, 1877), Ctenus
medius (Keyserling, 1891), Phoneutria
nigriventer (Keyserling, 1891), Viracucha
andicola (Simon, 1906), and Enoploctenus
cyclothorax (Philip Bertkau, 1880), from Brazil. All species presented a 2n♂ = 28 except for V.
andicola, which showed 2n♂ = 29. Analysis of segregation and behavior of sex chromosomes during male meiosis showed a sex chromosome system of the type X1X20 in species with 28 chromosomes and X1X2X30 in V.
andicola. C banding stained with fluorochromes CMA3 and DAPI revealed two distributions patterns of GC-rich heterochromatin: (i) in terminal regions of most chromosomes, as presented in C.
medius, P.
nigriventer, E.
cyclothorax and V.
andicola and (ii) in interstitial regions of most chromosomes, in addition to terminal regions, as observed for C.
ornatus. The population of Ubatuba (São Paulo State) of this same species displayed an additional accumulation of GC-rich heterochromatin in one bivalent. Fluorescent in situ hybridization revealed that this bivalent corresponded to the NOR-bearing chromosome pair. All analyzed species have one bivalent with 18S rDNA site, except P.
nigriventer, which has three bivalents with 18S rDNA site. Karyotypes of two species, C.
medius and E.
cyclothorax, are described for the first time. The latter species is the first karyotyped representative of the subfamily Acantheinae. Finally, 18S rDNA probe is used for the first time in Ctenidae at the present study.