Pholcidae is the most diverse family among haplogyne spiders but only 15 species have been analyzed cytogenetically. These studies revealed that the diploid number varies from 2n  =  15 to 2n  =  32, that there are three types of sex chromosome systems in males (X, X₁X₂ and X₁X₂Y), and that the chromosomes are predominantly biarmed. Within the genus Mesabolivar, only Mesabolivar luteus (Keyserling 1891) has been karyotyped, and it showed 2n  =  15  =  14 X, with all chromosomes being metacentric. In the present work, we characterize the mitotic and meiotic chromosomes of Mesabolivar brasiliensis (Moenkhaus 1898) and Mesabolivar cyaneotaeniatus (Keyserling 1891). Male mitotic metaphases of the two species showed the diploid number 2n  =  17  =  16 X; oogonial cells of M. brasiliensis showed 2n  =  18  =  16 XX. In both species, the chromosomes were exclusively biarmed, and the X chromosome was the largest element of the karyotype. Diplotene spermatocytes of the two species exhibited 8II X and the occurrence of only one terminal or interstitial chiasma per bivalent. In M. cyaneotaeniaus, metaphases II with n  =  9  =  8 X and n  =  8 were found, indicating the regular segregation of all chromosomes during meiosis I. Mitotic metaphases of M. brasiliensis stained with CMA₃/DA/DAPI revealed GC-rich chromatin in the terminal region of almost all autosomes, especially in pair 2. An earlier revision of the New World pholcids grouped M. brasiliensis and M. cyaneotaeniatus in a “southern group” and placed M. luteus in a “miscellaneous group.” A molecular study showed a closer relationship between M. brasiliensis and M. cyaneotaeniatus than between M. luteus and either of these two species. The 2n  =  17 found in M. brasiliensis and M. cyaneotaeniatus corroborates this hypothesis, given that M. luteus has a diploid number of 2n  =  15.