Species of Polybetes are known exclusively from South America. Currently there are 13 described species, 9 occurring in Argentina. Cytogenetic studies in spiders are scarce; the cytogenetics of only about 1% of nearly 39,500 described species are known. Within the Sparassidae, 38 species out of 1,009 (< 4%) have been cytogenetically analyzed; the most frequent chromosome number is 2n = 43/46 (male/female), n = 20 X₁X₂X₃, present in almost half of the species studied. Female diploid chromosome number is only known for four species: Heteropoda venatoria (Linnaeus 1767) (2n = 44); Pediana regina (L. Koch 1875), Isopeda sp. and Olios sp. (2n = 46). Within the genus Polybetes, only P. pythagoricus (Holmberg 1875) had been previuosly cytogenetically analyzed. In the present work, the karyotype, heterochromatin content and distribution, and silver stained nucleolus-organizer regions of P. pythagoricus, P. rapidus (Keyserling 1880) and P. punctulatus Mello-Leitão 1944 are described and compared. In P. pythagoricus the identification of the chromosome pairs by means of G-banding is also performed. Females of the three species show a chromosome complement of 44 telocentric chromosomes, with a similar karyotype. Males of P. pythagoricus show 42 telocentric chromosomes, the two sex chromosomes being the largest and of different size. In the three species, two pairs of telomeric NORs and small pericentromeric positive C-bands in all chromosomes were detected. This C-banding pattern seems to be characteristic of spiders. Comparative analysis of chromosome complements in Sparassidae indicates that 2n = 42/44 (X₁X₂0/X₁X₁X₂X₂) (male/female) may represent the ancestral karyotype for Polybetes.