Dactylopius opuntiae (Cockerell) (Hemiptera: Dactylopiidae) is a new invasive insect in our area, and the most destructive insect on cactus species. It is often referred to as prickly pear cochineal and produces large economic losses all over the globe. This research investigates the molecular genetics of D. opuntiae. Molecular studies exploring the evolutionary relationships of recently established and naturally occurring D. opuntiae populations are crucial in determining the routes of entrance. Here, the sequence variation in the mtCOI, 12S and 18S gene markers from specimens isolated from the Palestinian territories in 2023 were assessed to gain greater insight into the variations in DNA within and between Palestinian D. opuntiae populations. The results showed the presence of 12, 18, and 12 haplotypes for COI, 12S, and 18S gene markers, respectively. Furthermore, the results indicated low nucleotide diversity (π) (0.00133 for COI, 0.00258 for 12S, and 0.00085 for 18S) and high haplotype diversity (Hd) within haplotype phylogenetic trees (0.0.563 for COI, 0.628 for 12S, and 0.425 for 18S). Because the sequences deposited in GenBank are from few countries, it was difficult to track the origin of Palestinian sequences with COI, 12S, and 18S sequences deposited in GenBank from few areas of the world. In conclusion, the Palestinian D. opuntiae population exhibited the highest Hd and lowest π based on investigated gene markers. In addition, negative results for neutral tests indicated that a high number of uncommon mutations found in the populations, which may suggest new colonization events and increase in population number.