Historically Buthus occitanus (Amoreux 1789) was recognized as the sole species of the genus present in the Iberian Peninsula, but recent morphological studies have identified and named two additional species. In addition, molecular data on the Moroccan fauna has shed light on the diversity within the genus. More species have since been described from North Africa, where diversity within the genus is highest. In this study we assessed the genetic diversity within specimens of Buthus Leach 1815 from across the Iberian Peninsula using cytochrome oxidase 1 (CO1) mitochondrial DNA sequences. The known range of B. ibericus Lourenço & Vachon 2004 was greatly expanded, with the species widespread in most of the western part of the Iberian Peninsula. Five distinct mtDNA lineages were found within Buthus from the Iberian Peninsula, two of which were reported for the first time in this study. However, both B. ibericus and B. occitanus included highly divergent lineages and thus further studies are needed to fully comprehend the taxonomy of Buthus from this region.