Platypus cylindrus is an important wood borer of cork oak trees (Quercus suber) in the Mediterranean region, namely Portugal, Morocco and Algeria where its presence has drastically increased in the past few decades. On the contrary, the insect is not a relevant pest in Tunisia. The aim of this work is to analyze morphological and genetic differences among Tunisian and Portuguese populations in order to understand their role in the diverse population dynamics (e.g., aggressiveness) of the insect. The information could be used as a novel tool to implement protective measures. Insects were collected from cork oak stands in Tunisia (Ain Beya, Babouch and Mzara) and Portugal (Chamusca and Crato). Morphological traits of female and male mycangial pits were determined, using scanning electron microscopy but no significant differences were found. Genetic differences were analyzed using nuclear (internal simple sequence repeat polymerase chain reaction) and mitochondrial (cytochrome oxidase I (COI)) molecular markers. The results showed a very low level of intraspecific polymorphism and genetic diversity. The alignment of COI sequences showed high percentage of identical sites (99%) indicating a very low variation in nucleotide composition. Other variables related with the ecology of the insect and its associated fungi must be studied for a better understanding of the differences in the insect population's dynamic in Mediterranean countries.