Neobenedenia spp. are harmful ectoparasites that can cause disease and mortality in aquaculture systems. The identification of Neobenedenia spp. is difficult using only morphological characteristics; therefore, molecular techniques represent a very useful tool to differentiate species, as recently demonstrated for Neobenedenia melleni and Neobenedenia girellae. In line with this, DNA barcoding (a region of the mitochondrial cytochrome c oxidase subunit I (COI)) can provide strong resolution at the species level for different parasite groups. Neobenedenia spp. have been reported in captive Seriola species along the western Pacific Ocean and in several wild fish species from the Chilean coast (South-Eastern Pacific, SEP) but has never been previously reported in wild and farmed Seriola Ialandi from SEP. During routine sampling in 2015 and 2017, specimens of Neobenedenia sp. were recorded, for the first time, in S. Ialandi cultured in a hatchery. In the present study, we used mitochondrial DNA (the COI gene) and nuclear DNA (28S LSU rDNA) to support the specific identification of these monogeneans. Then, we evaluated the potential source of infestation of Neobenedenia sp. by comparing COI sequences and morphometry of monogenean specimens from five abundant wild littoral fish species collected by local fishermen from the SEP. Parasites were sorted and fixed in 70% and absolute ethanol for taxonomic identification, morphometric measurements and molecular analysis. In total, 58 COI sequences of Neobenedenia sp. were identified; each of these was 641 bp in length. The least genetic distance of Neobenedenia spp. was recorded between Neobenedenia sp. from S. Ialandi and Cheilodactylus variegatus (0.2%-1.2%) and between sequences from S. Ialandi and Aplodactylus punctatus (0.4%-1.4%). Neobenedenia specimens from Paralabrax humeralis were the most genetically distant with respect to the other specimens used for comparison. Molecular analysis determined that Neobenedenia species from cultured S. Ialandi and littoral fish species from the SEP are genetically distinct from N. melleni and N. girellae. In addition, our results suggest that there are at least two species of Neobenedenia in wild fish from the Chilean coast Furthermore, the morphometry of Neobenedenia specimens differed among different host species. Finally, given the higher genetic similarity between Neobenedenia sp. from the most abundant littoral fish species (C. variegatus and A. punctatus) and those parasites from S. lalandi, we suggest that these wild host fishes were important in transmitting Neobenedenia sp. to cultured S. Ialandi in a hatchery.