The present study elevates the number of cytogenetically analyzed ctenid species and genera from two to eight and six, respectively, presenting comparisons between chromosomal data obtained and the phylogenetic hypothesis proposed in the literature. Six ctenid species presented 13 autosomal pairs, exhibiting either X₁X₂O (Ctenus ornatus, Ctenus sp., Parabatinga brevipes and Phoneutria nigriventer) or X₁X₂X₃O sex chromosome systems (Nothroctenus sp. and Viracucha andicola). Asthenoctenus borellii showed 2n ♂ = 20 X₁X₂O. In all species, the chromosomes were telocentric. Some cells of one C. ornatus specimen exhibited one extra chromosome that, considering the behavioral similarities between the two chromosomes, can be considered to be supernumerary, derived from or giving rise to a sex chromosome. Silver impregnation revealed nucleolar organizer regions on one autosomal pair of C. ornatus and P. nigriventer (Cteninae) and two pairs of V. andicola (Acanthocteninae). Chromosomal data suggests that the X₁X₂X₃O system arose several times in the evolution of entelegyne spiders, and that conversion of an X₁X₂O system into an X₁X₂X₃O system and vice-versa has been a relatively common event in spiders. All the chromosomal data corroborate the close relationship between Ctenus and Phoneutria, the placement of P. brevipes within Cteninae, the placement of Anahita in a separated branch within Cteninae, and the inclusion of A. borellii in a distinct group within the ctenids (Viridasiinae), all of which are as proposed by phylogenetic hypotheses available in the literature.