Glyphorynchus spirurus (GSP), also called the Wedge-billed Woodcreeper (Furnariidae) has an extensive distribution in the Americas, including the Atlantic coast of Brazil. Nevertheless, there is no information about its karyotype or genome organization. To contribute to the knowledge of chromosomal evolution in Passeriformes we analysed the karyotype of Glyphorynchus spirurus by classic and molecular cytogenetics methods. We show that Glyphorynchus spirurus has a 2n = 80 karyotype with a fundamental number (FN) of 84, similar to the avian putative ancestral karyotype (PAK). Glyphorynchus spirurus pair 1 was heteromorphic in the Tapajós population whereby the short arms varied in sizes, possibly due to a pericentric inversion, as described in other Furnariidae birds. FISH with the Histone H5 probe revealed a signal in the pericentromeric region of G. spirurus chromosome 5 and rDNA 18S showed interstitial signal in GSP-1. Chromosome painting with Gallus gallus (GGA) macrochromosomes 1-9 probes showed disruption of chromosome syntenies of GGA-1, 2 and 4 by fission in Glyphorynchus spirurus. Our results confirm that the GGA1 centric fission is a synapomorphic character for the phylogenetic branch composed of Strigiformes, Passeriformes, Columbiformes and Falconiformes. On the other hand, the GGA-2 fission is reported here for the first time in Passeriformes. Chromosome painting with BOE whole chromosome probes confirmed these rearrangements in Glyphorynchus spirurus revealed by Gallus gallus 1-9 probes, in addition to enabling the establishment of genome-wide homology map.