The tick species that comprise the Amblyomma maculatum group (Acari: Ixodidae) are widely distributed in the Nearctic and Neotropical regions, ranging from the United States of America (USA) to Argentina. This group includes three species: Amblyomma maculatum, Amblyomma tigrinum, and Amblyomma triste, which parasitize a high number of vertebrates, including domestic mammals and humans, where they are the main vectors of Rickettsia parkeri s.s. The identification, distribution, and validity of the three species within the group have been controversial and continue to raise questions. Previous studies have mostly focused on the analysis of partial nuclear and mitochondrial genes, highlighting the need for new integrative analyses that contribute to clarifying their systematics and ecology. In this investigation, we obtained the first mitochondrial genome of A. triste (14,808 bp), which, upon comparison with the reported mitochondrial genomes of A. maculatum, exhibits a genetic distance of 4.2%, providing new evidence for the validity of the former species. The order, composition, and structure of the mitogenome of A. triste are consistent with the characteristics reported for most Metastriata including taxa within Amblyomma, that count with several mitochondrial genomes published in the last few years. The attributes of the mitogenomes, such as the absence of paralogs and a small genome, confirm the utility in studies involving complexes of cryptic species as it also serves as a robust tool for inferring phylogenies. Furthermore, we reported new molecular markers at the mitochondrial level which can be used in phylogenetic studies for other tick species, especially those with controversial or challenging taxonomy. We highlight the need of sequencing the mitochondrial genome of A. tigrinum, which is part of the A. maculatum group, as well as experimental crosses of populations from different places in the Americas to obtain additional evidence for species recognition.