The spatial genetic structure (and gene flow) of parasites with complex life cycles, such as digeneans, has been attributed mainly to the dispersion ability of the most mobile host, which most often corresponds to the definitive host (DH). In this study, we compared the genetic structure and diversity of adult Neolebouria georgenascimentoi in two fish species (DHs) that are extensively distributed along the Southeastern Pacific (SEP). The analysis was based on the cytochrome oxidase subunit I gene sequences of parasites collected between 23°S and 45°S. In total, 202 sequences of N. georgenascimentoi in Pinguipes chilensis isolated from 9 sites and 136 sequences of Prolatilus jugularis from 5 sites were analyzed. Our results showed that N. georgenascimentoi is a species complex that includes three different parasite species; however, in this study, only group 1 and 2 found in P. chilensis and P. jugularis, respectively, were studied because they are widely distributed along the coastline. Group 1 parasites had two common haplotypes with wide distribution and unique haplotypes in northern sites. Group 2 had only one common haplotype with wide distribution and a large number of unique haplotypes with greater genetic diversity. Both groups have experienced recent population expansion. Only group 1 exhibited a genetic structure that was mainly associated with a biogeographic break at approximately 30°S along the SEP. Our finding suggests that host access to different prey (=intermediate hosts) could affect the genetic structure of the parasite complex discovered here. Consequently, difference between these patterns suggests that factors other than DH dispersal are involved in the genetic structure of autogenic parasites.