The tyrannids are one of the most diverse groups of birds in the world, and the most numerous suboscine family in the Neotropics. Reflecting such diversity, many taxonomic issues arise in this group, mainly due to morphological similarities, even among phylogenetically distant species. Other issues appear at higher taxonomic levels, mostly brought up by genetic studies, making systematics a rather inconclusive issue. This study looks into the use of DNA barcodes method to discriminate and identify Tyrannidae species occurring in the Atlantic Forest and Cerrado biomes of Brazil. We analysed 266 individuals of 71 tyrant-flycatcher species from different geographical locations by sequencing 542 bp of the mtDNA COI gene. The great majority of the analysed species showed exclusive haplotypes, usually displaying low intraspecific diversity and high interspecific divergence. Only Casiornis fuscus and Casiornis rufus, suggested in some studies to belong to a single species, could not be phylogenetically separated. High intraspecific diversity was observed among Elaenia obscura individuals, which can suggest the existence of cryptic species in this taxon. The same was also observed for Suiriri suiriri, considered by some authors to comprise at least two species, and by others to be divided into three subspecies. Additionally, the use of sequences from voucher specimens allowed us to correct four misidentifications that had happened in the field. Our findings suggest a great power of the COI barcodes to discriminate species of the Tyrannidae family that are found in Brazil.