The recent description of Amoebic Gill Disease (AGD) and Neoparamoeba perurans in Atlantic salmon (Salmo salar) farmed in Chile has necessitated the development of more reliable and sensitive diagnostic tests. Final diagnosis of infection is normally confirmed by histology. However, the correlation between gross gill lesions and histological lesions is generally unclear. In the current study, moderate concordance level (k=0.5319) between gross pathology and histology was observed. The sensitivity and specificity of gross pathology was 77.91% and 71.05%, respectively. Neoparamoeba spp. are considered morphologically indistinguishable therefore by using histopathology limits the capacity to characterise the causative agent and it can be time consuming. We developed a PCR assay to amplify the N. perurans 18S rRNA gene from gill clinical samples of AGD-affected fish. High concordance level (k=0.95) between PCR and histological examination was observed. The sensitivity and specificity of PCR assay was 94.64% and 97.06%, respectively. The PCR-based assay provides a rapid tool that will be useful to the diagnostic routine for AGD in Chile.