In 2020, multiple lionfish (Pterois spp.) records along the equatorial Southwestern (SW) Atlantic revealed a new expansion of these potentially damaging invasive populations, which could impact over 3500 km of Brazilian coastline over the next few years, as well as unique ecosystems and marine protected areas in its path. To assess the taxonomic status, invasion route, and correlation with other centres of distribution, we investigated the genetic diversity patterns of lionfish caught in 2022 at the Amazonia, Northeastern Brazil, and Fernando de Noronha and Rocas Atoll ecoregions, using two molecular markers, the mitochondrial COI and the nuclear S7 RP1. The data indicate that all studied lionfish belong to what is generally accepted as P. volitans, and share the same genetic signature as lionfish present in the Caribbean Sea. The shared haplotypes and alleles indicate that the SW Atlantic invasion derives from an active movement of adult individuals from the Caribbean Sea into the Brazilian coast. The Amazon mesophotic reefs likely served as a stepping-stone to overcome the biogeographical barrier represented by the Amazon-Orinoco River plume. New alleles found for S7 RP1 suggest the onset of local genetic diversification, heightening the environmental risks as this bioinvasion heads towards other South Atlantic ecoregions.