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Green tree frogs of the genus Aplastodiscus occur in the Atlantic Forest and Cerrado biomes of South America. The genus comprises 15 medium-sized species placed in three species groups diagnosed mainly by cloacal morphology. A phylogenetic analysis was conducted to: (1) test the monophyly of these species groups; (2) explore the phylogenetic relationships among putative species; and (3) investigate species boundaries. The dataset included eight mitochondrial and nuclear gene fragments for up to 6642 bp per specimen. The results strongly support the monophyly of Aplastodiscus and of the A. albofrenatus and A. perviridis groups. Aplastodiscus sibilatus is the sister taxon of all other species of Aplastodiscus, making the A. albosignatus Group non-monophyletic as currently defined. At least six unnamed species are recognized for Aplastodiscus, increasing the diversity of the genus by 40%. A fourth species group, the A. sibilatus Group is recognized. Aplastodiscus musicus is transferred from the A. albofrenatus Group to the A. albosignatus Group, and A. callipygius is considered a junior synonym of A. albosignatus. Characters related to external cloacal morphology reveal an interesting evolutionary pattern of parallelisms and reversions, suggesting an undocumented level of complexity. We analyze, in light of our phylogenetic results, the evolution of reproductive biology and chromosome morphology in Aplastodiscus., (Copyright © 2015 Elsevier Inc. All rights reserved.)