The present study aimed to investigate the diversity of Macrochelidae species associated with sheep feces at the Instituto de Zootecnia of Nova Odessa, São Paulo, Brazil, using an integrative taxonomic approach. Twenty-seven sheep feces samples were collected. Mites were extracted using an adapted Berlese–Tullgren funnel, followed by morphological identification through light microscopy and scanning electron microscopy. Molecular analysis included non-destructive DNA extraction, amplification, and sequencing of the COI gene region. Phylogenetic analyses were conducted using the maximum likelihood method, and genetic distances were estimated from the sequences obtained. Fifteen species belonging to four genera were identified: Macrocheles (Macrocheles dimidiatus, Macrocheles insignitus, Macrocheles mammifer, Macrocheles merdarius, Macrocheles muscaedomesticae, Macrocheles peniculatus, Macrocheles robustulus, Macrocheles roquensis, Macrocheles subadius, and Macrocheles sp.), Holostaspella (Holostaspella bifoliata and Holostaspella berlesei), Glyptholaspis (Glyptholaspis confusa and Glyptholaspis fimicola), and Allogynaspis (Allogynaspis bolivari), totaling 1489 individuals. Two species, H. berleseiand G. fimicola, were recorded for the first time in Brazil. Of the 15 species identified, 8 were sequenced. Phylogenetic analyses confirmed the monophyly of Macrochelidae, with good branch support, with 50% of the nodes showing bootstrap values above 95. Interspecific genetic distance ranged from 0.01571 to 0.2933, indicating broad divergence among the species analyzed. Comparisons between the species sequenced in this study and sequences available in databases showed that M. robustulus and M. scutatiformis had the greatest genetic proximity, whereas M. merdarius and Longicheles sp. exhibited the greatest divergence. Among the species analyzed in this study, intraspecific genetic variation ranged from 0.168 to 0.2673, with Macrocheles dimidiatus and H. bifoliata being the closest, and M. merdarius and G. fimicola the most distant. The results indicate high Macrochelidae diversity in sheep feces and demonstrate that integrative taxonomy is an effective tool for delimiting and differentiating species, including among morphologically similar taxa.