Abstract The family Characidae is the most diverse group of fishes in the Neotropics with challenging systematics. The three genera Carlana, Parastremma, and Rhoadsia, formerly considered the subfamily Rhoadsiinae, are now included in the subfamily Stethaprioninae. Previous phylogenetic analyses did not include all genera of Rhoadsiinae, specifically Parastremma. Here, we estimated the phylogenetic relationships and divergence times of the genera of Rhoadsiinae (the Rhoadsia clade) relative to the most representative genera of the Characidae. We used six molecular markers from the mitochondrial and nuclear genome to estimate the phylogeny and divergence times. We confirmed the monophyly of the Rhoadsia clade. Furthermore, we estimated that the Central American genus Carlana and the western Colombian genus Parastremma diverged approximately 13 Mya (95% HPD 8.36–18.11), consistent with the early-closure estimates of the Isthmus of Panama (~15 Mya). The genus Rhoadsia, endemic to Western Ecuador and Northern Peru, was estimated to originate at around 20 Mya (95% HPD 14.35–25.43), consistent with the Andean uplift (~20 Mya).