The karyotype of a freshwater population of Diplodon chilensis located in La Poza area of the Villarrica Lake (39º18'S; 72º05'W), Southern Chile, was studied. The chromosomes were obtained by squash of cleaving embryos, previously treated with colchicine and fixed in ethanol-acetic acid 3:1 at 4ºC and stained using the Feulgen reaction. Diplodon chilensis showed a diploid karyotype of 2n = 34, with metacentric and submetacentric chromosomes. This work is the first approach to cytogenetic characterization of Chilean populations of this species.