Spiders represent a fascinating group for studying chromosomal evolution due to their dynamic karyotypes, which reveal significant differences even between closely related taxa. Lycosidae, one of the most species-rich families of spiders, has its major relationships well stablished, though minor interspecific relationships remain poorly understood. Here, we analyzed chromosomal data from four species belonging to Aglaoctenus and Diapontia, two genera within the subfamily Sosippinae, to discuss the evolution of chromosomal traits within this lineage. For karyological analysis, we employed Giemsa staining, C-banding, Ag-NOR staining, and 28S rDNA FISH to identify key chromosomal characteristics and the distribution of repetitive elements. Both A. oblongus and D. uruguayensis exhibited 2n♂=28, X1X2, with acro/telocentric morphology, hypothesized to represent the ancestral condition for Lycosoidea. However, regarding the 28S rDNA chromosome mapping, these species revealed an unusually high number of loci with eight chromosome clusters in A. oblongus and a polymorphic condition in D. uruguayensis (10–14 signals), underscoring the utility of additional techniques for detecting specific chromosomal regions. Interestingly, A. castaneus and A. lagotis displayed a more derived karyotype, characterized by 2 n♂= 14, X1X2 and acro/telocentric morphology, with the sex chromosomes (X1 and X2) smaller than the autosomes. Taken together, these data suggest that, following the diversification of Aglaoctenus, a significant reduction in diploid number occurred, affecting only the autosomes. These findings encourage further cytogenetic research within Sosippinae and support the use of these data in future phylogenetic studies.