Diphyllobothriosis in fish from freshwater ecosystems in southern Chile was first reported in 1949. Infection by plerocercoids of Diphyllobothrium latum and Diphyllobothrium dendriticum occurs in introduced trout (Oncorhynchus mykiss) and native fish. We determined the prevalence, mean intensity, and mean abundance of seasonal infection and tissue damage produced by Diphyllobothrium spp. in native fish (Percichthys trucha, Odontesthes mauleanum, and Basilichthys australis) and introduced trout (O. mykiss) from Lake Panguipulli, Chile. Prevalence, mean intensity, and mean abundance of D. latum infection were significantly greater in trout than they were in native fish. Prevalence and mean abundance were similar in O. mauleanum and P. trucha, but they were greater than those in B. australis. Prevalence and abundance were similar among seasons between sexes for the four hosts. For all host species, except P. trucha, there was a statistically significant positive correlation between host length and the abundance of plerocercoids. Infections in muscle tissue were present in 61% of trout compared with 23% in O. mauleanum and 12% in P. trucha, suggesting a greater risk for human infection when consuming trout. In general, prevalence of infection by D. dendriticum was lower than was D. latum prevalence. Encapsulation of plerocercoids was common and severe in 71% of the trout examined. Only slight encapsulation of plerocercoids was found in the native O. mauleanum, and no encapsulation was observed in P. trucha or B. australis. The greater concentration of plerocercoids in the walls of the digestive tract of trout suggests a more-rapid immune response in trout than in native fish. The low frequency of encapsulation of plerocercoids in native fish would mean greater tissue damage in the natives than that observed in the trout because they are free to migrate among the viscera, potentially endangering these native fish populations in regions where Diphyllobothrium spp. are endemic.