Cytogenetic analyses were performed on several populations of the Central American tree frog Agalychnis callidryas, using conventional methods and banding techniques. The karyotype of this species is distinguished by an inversion polymorphism in chromosome 9, which is either submetacentric or telocentric. The populations examined are in Hardy-Weinberg equilibrium with respect to the two alternative morphs of chromosome 9. This is the first report of the occurrence of an intrapopulational chromosomal inversion polymorphism in the order Anura. In male meiosis, the two chromosomes 9 form a bivalent exhibiting a ring-like pairing configuration with terminal chiasmata in both arms, regardless of whether the paired homologs are heteromorphic or homomorphic. Furthermore, individual specimens of A. callidryas exhibit one or two unexpected 18S + 28S ribosomal RNA gene clusters, in addition to the standard nucleolus organizers. The chromosomal localization of these extra nucleolus organizers is identical in all metaphases from the same specimen and shows a specific intraindividual pattern. The karyotype evolution in the phyllomedusine hylids, the structure of the various classes of heterochromatin, and the occurrence and possible origin of the rare inversion polymorphisms and multiple nucleolus organizers in A. callidryas and a few other amphibian species are discussed.