The family Filistatidae is considered sister to Synspermiata or sister to Hypochilidae. Cytogenetic knowledge of this family could be useful for understanding the mechanism of chromosome evolution that has occurred within the group. In this work, two filistatid species belonging to distinct subfamilies, Kukulcania hibernalis (Hentz, 1842) (Filistatinae) and Misionella mendensis (Mello-Leitão, 1920) (Prithinae), were investigated using standard and differential chromosome staining. Analysis of mitotic and meiotic cells revealed the diploid 2n♂ = 25 for K. hibernalis and 2n♂ = 21 for M. mendensis. Both species exhibited a sex chromosome system of the X₁X₂Y type and metacentric/submetacentric chromosomes. In prophase I cells, the sex chromosomes were in a trivalent configuration with all elements associated without chiasma through their terminal regions. Both species revealed six nucleolar organizer regions on the terminal region of three autosomal pairs. In K. hibernalis, constitutive heterochromatin was located mainly in the terminal regions of autosomes and sex chromosomes while in M. mendensis, the heterochromatin occurred in the pericentromeric region of all chromosomes. Despite the scarcity of cytogenetic information for Filistatidae, the available results show the occurrence of high variability in the diploid number but with the maintenance of the X₁X₂Y sex chromosome system. Additionally, the karyotype differentiation in the species of this family seems to have involved not only the number of autosomes but also specific chromosomal sites, such as the constitutive heterochromatic regions.