The Cuckoos have a long history of difficult classification. The species of this order have
been the subject of several studies based on osteology, behavior, ecology, morphology and
molecular data. Despite this, the relationship between Cuculiformes and species of other
orders remains controversial. In this work, two species of Cuculidae, Guira guira (Gmelin,
1788) and Piaya cayana (Linnaeus, 1766), were analyzed by means of comparative chromosome painting in order to study the chromosome evolution of this group and to undertake
the first chromosome mapping of these species. Our results demonstrate high chromosomal
diversity, with 2n = 76 in G. guira, with fission and fusion events involving ancestral syntenies, while P. cayana presented only fissions, which were responsible for the high diploid
number of 2n = 90. Interestingly, there were no chromosomal rearrangements in common
between these species. Our results, based on Giemsa staining, were compared with previous data for other cuckoos and also with taxa proposed as sister-groups of Cuculiformes
(Otidiformes, Musophagiformes and Opisthocomiformes). Cytogenetic comparisons demonstrated that cuckoo species can be divided into at least three major groups. In addition,
we found no evidence to place Cuculiformes close to the groups proposed previously as sister-groups.
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