Different pathways of propagation and dispersal of non-native species into new environments may have contrasting demographic and genetic impacts on established populations. Repeated introductions of rainbow trout (Oncorhynchus mykiss) to Chile in South America, initially through stocking and later through aquaculture escapes, provide a unique setting to contrast these two pathways. Using a panel of single nucleotide polymorphisms, we found contrasting genetic metrics and patterns among naturalized trout in Lake Llanquihue, Chile’s largest producer of salmonid smolts for nearly 50 years, and Lake Todos Los Santos (TLS), a reference lake where aquaculture has been prohibited by law. Trout from Lake Llanquihue showed higher genetic diversity, weaker genetic structure and larger estimates for the effective number of breeders (Nb) than trout from Lake TLS. Trout from Lake TLS were divergent from Lake Llanquihue, and showed marked genetic structure and a significant isolation-by-distance pattern consistent with secondary contact between documented and undocumented stocking events in opposite shores of the lake. Multiple factors, including differences in propagule pressure, origin of donor populations, lake geomorphology, and habitat quality or quantity, may concomitantly explain contrasting genetic metrics and patterns for trout between lakes. We also discussed violations of assumptions that may lead to overestimating Nb . We contend that high propagule pressure from aquaculture may not only increase genetic diversity and Nb via demographic effects and admixture, but also may impact the evolution of genetic structure and increase gene flow, consistent with findings from artificially propagated salmonid populations in their native and naturalized ranges.