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Background: Amblyomma spp. ticks, known for their long mouthparts, bright ornate appearance and aggressive hunting behaviour, are vectors of a number of important pathogens. In southern Africa, 17 Amblyomma spp. are currently documented. Of these species, Amblyomma hebraeum and Amblyomma variegatum have been well studied due to their wide geographical range and their status as competent vectors of pathogens that are of veterinary and medical importance. Studies on other Amblyomma spp. in southern Africa have been neglected, fostering ongoing debates on the validity of certain species such as Amblyomma pomposum. This study investigated the inter- and intra-species variation of Amblyomma ticks collected in southern Africa, focusing on resolving the dispute about A. pomposum and A. variegatum being distinct species. Methods: Four Amblyomma tick species were collected from Angola, Mozambique, South Africa, Zambia and Zimbabwe, and were identified morphologically as Amblyomma eburneum (208), A. hebraeum (4758), A. pomposum (191) and A. variegatum (2577) using identification keys. Gene amplification targeting the 12S and 16S rRNA, cytochrome oxidase I, cytochrome B and internal transcribed spacer-2 genes was conducted for 204 ticks, for which varying success was achieved during amplification for each of the markers. Maximum likelihood analyses were performed in IQ-TREE. Results: The phylogenetic topologies and ABGD analyses of each individual gene clustered A. pomposum within the A. variegatum clade, while clearly separating A. eburneum and A. hebraeum from all other species. None of the genetic markers indicated intraspecific structuring on the basis of geographical origin, despite great distances between sampling sites. Conclusion: Our study concludes that there is insufficient molecular evidence to differentiate A. pomposum and A. variegatum from each other. We highlight the need for whole mitochondrial genome sequencing of these two species to resolve the ongoing controversies. Furthermore, we propose mating and hybrid viability studies between the two species to confirm their reproductive isolation. [ABSTRACT FROM AUTHOR]