Five species of copepod crustaceans from four lakes in southern Chile were experimentally exposed to coracidia of Diphyllobothrium latum. Diphyllobothrium latum developed into mature procercoids in the calanoid copepods Boeckella gracilis and Diaptomus diabolicus, but only developed into the initial stage in the cyclopoid copepod Mesocyclops longisetus. The cyclopoid copepods Tropocyclops prasinus and Metacyclops mendocinus were not susceptible to the infection. In our experimental infections we obtained a prevalence of 26.8% in B. gracilis from Lake Malleco, 55.9% in D. diabolicus from Lake Rapel, 84.3% in D. diabolicus from Lake Panguipulli, and 7.7–22.6% for M. longisetus from Lake Panguipulli. The mean and maximum intensity were observed in D. diabolicus from Lake Panguipulli. We conclude that D. diabolicus is probably the primary intermediate host of D. latum because it is the most widespread copepod in southern Chile.