The Arabian rhinoceros beetle, Oryctes agamemnon arabicus (Burmeister, 1847), is an invasive species, introduced to Tunisia from the United Arab Emirates and causing serious damage to date palm trees in southern Tunisia. Considering the economic importance of this pest, it is necessary to understand the genetic diversity within and among its populations and investigate its dispersal mode. In the present study, the mitochondrial gene encoding the cytochrome oxidase subunit I (COI) was used to analyse the extent of genetic variation between samples of O. agamemnon arabicus, collected from geographically distant locations in Tunisia. A 505 bp DNA fragment, resulting from PCR amplification, was sequenced. Representative sequences were aligned, revealing nine polymorphic sites that identified eight haplotypes. Haplotype diversity (Hd) and Tajima's D neutral test were 0.707 and -0.043, respectively, indicating a low level of mtDNA variability. The calculated gene flow value was Nm = 0.47, indicating a high amount of gene flow occurring between populations from Tozeur and those from Kebili, although both departments are separated by the Chott-El-Jerid salt lake. The presence of common haplotypes between Tozeur and Kebili as well as the clustering pattern obtained by the NJ method confirmed this result. Based on these facts and considering the limited flight of O. agamemnon arabicus and its slow reproduction, it is likely that its expansion in Tunisia was facilitated mainly by human transportation of infected offshoots.