Aim  To create a molecular phylogenetic hypothesis for the closely related serranid genera Alphestes Bloch and Schneider and Dermatolepis Gill and assess the role of the Panamanian Isthmus in speciation within these reef fishes. Location  Tropical eastern Pacific, Caribbean, and Indian Oceans. Methods  Sequence data from one nuclear (TMO-4C4) and three mitochondrial genes (16S, 12S, and cytochrome b) were used in maximum parsimony and maximum likelihood analyses. Results  Here we show that previously hypothesized trans-isthmian geminate species are not each other's closest living relatives. Species of Alphestes Bloch and Schneider in the eastern Pacific are sister taxa indicating post-closure speciation. Within Dermatolepis Gill, we identify a sister group relationship between the Caribbean and western Indian Ocean species, a rarely reported biogeographic pattern. Based on sequence divergence, speciation among the three species of Dermatolepis was, however, nearly simultaneous around the time of the isthmian closure event. Main conclusions  Our molecular phylogenetic analysis of two closely related genera of reef fishes, each with presumed trans-isthmian geminates, cautions against the uncritical use of morphological similarity in identification of geminates, as well as the assumption that trans-isthmian sister groups date to the isthmian closure event. These findings suggest that in some instances incomplete sampling of species within a clade including putative geminates may lead to improper conclusions regarding the pattern and timing of speciation, as well as incorrect estimation of the rate at which evolution has proceeded.