The palearctic spider genus Mastigusa Menge, 1854 is characterized by a remarkable morphology and wide ecological variability, with free-living, cave dwelling and myrmecophile populations known. This genus has a long and tangled taxonomic history and was placed in different families in the past, all belonging to the “marronoid clade”, an informal grouping of families characterized by the lack of strong synapomorphies. Three species are currently recognized, but their identity and circumscription has been long debated. A molecular approach was never applied for trying to solve these uncertainties, and doubts still remain both about its phylogenetic placement and about the taxonomic status of the described species. For the first time the genus Mastigusa is included in a molecular phylogenetic analysis and strong support is found for its placement within the family Cybaeidae, in sister relationship with the genus Cryphoeca Thorell, 1870. An analysis of Mastigusa populations spanning across the distribution range of the genus identifies a high and previously overlooked genetic diversity, with six distinct genetic lineages showing a strong geographic pattern. Divergence times between Mastigusa and its sister genus and between the distinct Mastigusa lineages are estimated, and the groundwork is laid for a taxonomic revision of the species belonging to the genus.