Heroini constitute the second largest tribe of Neotropical cichlids and show their greatest diversity in Mesoamerica. Although heroine species are morphologically and ecologically very diverse, they were all historically assigned to one single genus, Cichlasoma that was never formally revised from a phylogenetic point of view. Here, we present the most comprehensive molecular phylogeny of the tribe Heroini to date, based on the complete DNA sequence of the mitochondrial gene cytochrome b, and the analysis of 204 individuals representing 91 species. Phylogenetic analyses did not support the monophyly of heroines because the genus Pterophyllum was placed as the sister group of all remaining heroines plus cichlasomatines. However, the recovered relative position of Pterophyllum was without strong statistical support. Within the remaining heroines, Hyspelecara and Hoplarchus are recovered with low support in a basal position with respect to a clade that includes Heros, Uaru, Mesonauta, and Symphysodon, and the circumamazonian (CAM) heroines. The first clade is restricted to South America. The largest clade of heroines, the CAM heroines, include more than 85% of the species within the tribe. This clade is mostly Mesoamerican, but also contains four species found in the Greater Antilles (Nandopsis), and three genera found in South America (the 'Heros' festae group, Australoheros, and Caquetaia). Up to eight major lineages can be recovered within the CAM heroines, but the phylogenetic relationships among them remain unresolved. Two large suprageneric groups can be distinguished, the amphilophines and the herichthyines. The amphilophines include Amphilophus, Archocentrus, Hypsophrys, Neetroplus, Parachromis, Petenia, and five additional unnamed genera (the 'Heros' istlanus group, the 'Amphilophus' calobrensis group, the 'Heros' urophthalmus group, the 'Heros' wesseli group, and the 'Heros' sieboldii group). The herichthyines include the crown-group herichthyines (Herichthys, Theraps, Vieja, and Paratheraps) and the genera Tomocichla, Herotilapia, and Thorichthys, together with three unnamed genera (the 'Heros' umbriferus group, the 'Heros' grammodes group, and the 'Heros' salvini group). Amphilophines are prevalent in southern Mesomerica south of the Motagua fault. Herichthyines have basal linages in Central America, whereas crown-group herichthyines and three related genera are found north from the Motagua fault. At least two independent origins are required to explain current Mesoamerican heroine distribution. Dispersal of heroines from South America into Mesoamerica was dated between 24 and 16 million years ago (MYA) based on geological calibrations and on standard fish mitochondrial cytochrome b rates, respectively. These datings cannot be reconciled with currently known geological evidence, and the existence of a connection between Central America and South America in the Miocene needs to be postulated in order to explain the origins of Mesoamerican heroine lineages. However, our datings agree with those estimated for the dispersal of other secondary freshwater fishes (Rivulidae, Synbranchus) into Mesoamerica, and predate the invasion of primary freshwater fishes by at least 10 myr.