Between May and November 2007, three marine Atlantic salmon farms around Chiloé Island, Chile, reported mortalities in which affected fish presented with Caligus rogercresseyi infections and gross gill lesions characteristic of amoebic gill disease (AGD). Histological examination of the gills from affected fish confirmed the presence of AGD lesions. Trophozoites possessing one or more endosymbiotic Perkinsela amoeba-like organisms (PLOs) were observed in association with hyperplastic tissue. Further analyses were undertaken using a combination of PCR and in situ hybridization and the trophozoites were identified as Neoparamoeba perurans. Thus, our data indicate that N. perurans is a causal agent of AGD in Chile. However, it is possible that AGD was not the single cause of mortalities in the epizootics investigated here. The exceptionally high level of co-infection with Caligus rogercresseyi (maximum mean intensity 34, prevalence 100%), could have contributed to the production losses.