The tree frog Aplastodiscus is a Neotropical taxon that encompasses 15 species in the Atlantic forest biome, with one isolated species in the Central Brazilian Cerrado. To date, only 8 species have been karyotyped, showing high levels of diploid number variation, which allowed clustering species in chromosome number groups: 2n = 24 (Aplastodiscus perviridis group), 2n = 22 (Aplastodiscus albofrenatus group), 2n = 20, and 2n = 18 (both within Aplastodiscus albosignatus group). This study aims to report karyotypic information on 4 species from the last 2 groups using classical and molecular cytogenetic techniques and hypothesize chromosomal evolutionary trends within the species groups. Aplastodiscus weygoldti showed 2n = 22; Ag-NOR and FISH 18S rDNA signals were located in the interstitial region of the short arms of chromosome pair 6. Aplastodiscus cavicola, Aplastodiscus sp. 4, and Aplastodiscus sp. 6 showed 2n = 18; Ag-NOR and FISH 18S rDNA bands were located in the terminal region of the long arm of chromosome pair 9. Our results support multiple and independent chromosome fusion events within Aplastodiscus, including a new chromosome fission event. Ag-NOR and FISH 18S rDNA patterns were restricted to the small chromosome pairs, similar to the other species within this genus, and confirm overall chromosome morphology conservation among the genera of Cophomantinae.