AbstractTheraphosidae is the most diversified family of mygalomorph spiders, commonly known as tarantulas. Two genera inhabit the Mediterranean region: Chaetopelma in the east and Ischnocolus mostly in the western part of the Basin. Their phylogenetic position and the validity of some Ischnocolus species remain unclear. We implemented a multilocus target approach to shed new light on the position of both genera and further integrated molecular data with additional lines of evidence (morphology and ecology) to explore species boundaries in western Mediterranean Ischnocolus. Our results reveal that Ischnocolus and Chaetopelma are not closely related. Chaetopelma formed a clade with the African subfamily Eumenophorinae and Ischnocolus was recovered in a clade comprising all remaining theraphosids. The western Mediterranean Ischnocolus comprises two deeply divergent clades that separated during the Early Miocene and differ in both morphology and lifestyle. We found molecular, morphological and ecological evidence to restore the name Ischnocolus mogadorensis and revalidate this species. We also uncovered distinct allopatric lineages in Ischnocolus elongatus. However, the lack of males, the uniform morphology of females and low within-clade support hampered the assessment of their status and boundaries. Finally, our data support that I. elongatus should be considered a senior synonym of Ischnocolus hancocki and Harpactirella insidiosa.