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Androctonus crassicauda is one of the medically important scorpion species occurring in the Middle East. In this study, molecular variation in mtDNA cytochrome oxidase subunit I (COI) gene within seven populations of A. crassicauda from four main ecological regions of Saudi Arabia was investigated. Scorpion specimens were collected from different eco-geographical regions. DNA was extracted, subsequently 700 bp fragments of COI gene were amplified using specific scorpion primers and sequenced. The obtained sequences were analyzed, and phylogenetic trees were constructed. The phylogenetic analysis showed high levels of genetic variation among A. crassicauda populations with the existence of three distinct lineages. Also, it supports the existence of two distinct populations of A. crassicauda in Saudi Arabia, which perhaps suggestive of a putative distinct species. Further morphological studies with additional specimens from the Arabian Peninsula may reveal possible undiscovered and cryptic species in the region.