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The phylogenetic position and taxonomic status of Rhynchocyclidae (Aves: Passeriformes) have been the subject of debate since their first description. In most models, Rhynchocyclidae represents a subfamily-level taxon placed within the Tyrant Flycatchers (Tyrannidae). Considering that this classification does not include cytotaxonomic characters, we tested the hypothesis that the chromosome organization of Rhynchocyclidae members differs from that of Tyrannidae. Hence, we selected two species, <i>Tolmomyias sulphurescens</i>, and <i>Pitangus sulphuratus</i>, representing Rhynchocyclidae and Tyrannidae, respectively. Results revealed a diploid number (2n) of 60 in <i>T</i>. <i>sulphurescens</i> and 2n = 80 in <i>P</i>. <i>sulphuratus</i>, indicating significant chromosomal differences. Chromosome mapping of <i>Gallus gallus</i> (GGA) and <i>Taeniopygia guttata</i> bacterial artificial chromosome (BAC) corresponding to chromosomes GGA1-28 (except 16) revealed that the genome evolution of <i>T</i>. <i>sulphurescens</i> involved extensive chromosome fusions of macrochromosomes and microchromosomes. On the other hand, <i>P</i>. <i>sulphuratus</i> retained the ancestral pattern of organization of macrochromosomes (except the centric fission involving GGA1) and microchromosomes. In conclusion, comparing our results with previous studies in Tyrant Flycatchers and allies indicates that <i>P</i>. <i>sulphuratus</i> has similar karyotypes to other Tyrannidae members. However, <i>T</i>. <i>sulphurescens</i> does not resemble the Tyrannidae family, reinforcing family status to the clade named Rhynchocyclidae.