Between April and June 2009, 1,075 feral rainbow trout from 10 different lakes involved with aquaculture activities in Los Lagos Region, Chile, were inspected for Diphyllobothrium species. All viscera and muscles of the fish were examined using stereomicroscopy; pyloric cecae and stomachs infected with plerocercoids were checked by histology and scanning electron microscopy. Plerocercoids of Diphyllobothrium dendriticum were confirmed by PCR and sequencing of COI and 18S rRNA ITS1 5.8S rRNA ITS2 genes for the first time in Chile. Overall prevalence of plerocercoids of D. dendriticum was 9.2% (99/1,075) in Los Lagos Region and 17.4% (99/570) for Chiloe Island. Plerocercoids were not detected in the continental lakes of the Los Lagos Region (Chapo, Rupanco, and Llanquihue). Tarahuín Lake exhibited a prevalence of 50.9% (81/159), Cucao Lake 5.1% (4/79), Natri Lake 4.7% (5/107), Huillinco Lake 3.6% (5/138), and San Antonio Lake 66.7% (4/6). Abundance was 1.1 plerocercoid larvae per fish (1,169 larvae/1,075 fish). All the plerocercoids were found encysted in the viscera of the fish. Plerocercoids were 10.9 ± 3 (7–16) mm long by 0.4 ± 0.2 (0.2–0.6) mm wide. The scolex was enlarged, with 2 bothria and a frontal pit. The body was covered with short capilliform filitriches, 4–6 mm long. The Chilean COI and 18SrRNA ITS1 5.8SrRNA ITS2 gene sequences indicated 96.34–96.52% and 99% similarity with D. dendriticum sequences, respectively. Diphyllobothrium dendriticum is reported for the first time in freshwater ecosystems as far as 43°S on Chiloe Island. These findings and previous reports of plerocercoids of Diphyllobothrium spp. in farmed rainbow trout at Tarahuín Lake support the putative life cycle of this parasite in lakes of southern Chile where there are aquaculture activities.