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•A description of the first mitochondrial genomes for Amblyomma cajennenses.s., A. mixtum, A. tonelliae, and A. patinoi; and new ones for A. sculptum.•Amblyomma cajennensecomplex phylogeny was recovered with strong statistical support.•Mitogenomes are stable in the Amblyomma cajennensecomplex and share the general features of Metastriata.•Mitochondrial genetic distances for the complex corroborate the clear differentiation between species.

Ticks of the Amblyomma maculatum(Acari: Ixodidae) group are represented by three species: Amblyomma maculatum, Amblyomma tigrinum, and Amblyomma triste. This group is widely distributed throughout the Nearctic and Neotropical regions, from the United States of America (USA) to Argentina. They parasitize several vertebrates, including domestic mammals and humans. Nonetheless, the identification and distribution of the species within the group have been controversial but can be solved with integrative studies on their taxonomy and ecology. In Colombia, a Neotropical country from which up to 58 tick species have been reported, only Amblyomma maculatumKoch, 1844 (sensu stricto - s.s.) - morphotype II – has been molecularly confirmed. Here, specimens of the A. maculatumgroup from three departments of Colombia were examined by morphological and molecular analyses. The results confirmed the wide distribution of A. maculatums.s. in Colombia. Furthermore, the presence of A. tristes.s. - morphotype I was confirmed for the first time at the molecular level, which broadens the distribution of this species in the American continent. Considering that both taxa are vectors of pathogens such as Rickettsia parkeris.s., additional taxonomic, reproductive compatibility and niche distribution studies are needed, as well as the application of new molecular markers due to the controversial taxonomy. This will allow us to explore cryptic diversity within the recently established putative groups.