Consisting of approximately 320 species, Macrocheles is the most widely distributed genus in the family Macrochelidae. Though some studies have focused on the description of Macrochelidae using molecular techniques (e.g., RAPD) and sequencing of some genes, the interspecies relationships within Macrocheles still remain uncertain. As such, in the present study, we examine all publicly available data in GenBank to explore the evolutionary relationships, divergence times, and amino acid variations within Macrocheles . Exploring the patterns of variation in the secondary protein structure shows high levels of conservation in the second and last helices, emphasizing their involvement in the energy metabolism function of the cytochrome oxidase subunit I enzyme. According to our phylogenetic analysis, all available Macrocheles species are clustered in a monophyletic group. However, in the reconstructed trees, we subdivided M. merdarius and M. willowae into two well‐supported intraspecific clades that are driven by geographic separation and host specificity. We also estimate the divergence time of selected species using calibration evidence from available fossils and previous studies. Thus, we estimate that the age of the Parasitiformes is 320.4 (273.3–384.3) Mya (Permian), and the Mesostigmata is 285.1 (270.8–286.4) Mya (Carboniferous), both with likely origins in the Paleozoic era. We also estimate that Macrocheles diverged from other Mesostigmata mites during the Mesozoic, approximately 222.9 Mya.