The mean karyotypes of four populations of Diplodon chilensis chilensis (Gray, 1828) (two lentic and two lotic) inhabiting the south of Chile are described and compared. Chromosomes were obtained by squash of cleaving embryos, previously treated with colchicine, fixed in ethanol-acetic acid 3:1 at 4°C, and stained using the Feulgen reaction. The karyotype of the four populations is 2n = 34, with metacentric and submetacentric chromosomes. In the population from the Peu-Peu stream, the centromeric position in two chromosome pairs (3 and 10) differs significantly from that in the corresponding pairs from the other populations. Karyotype findings of this study do not support the proposal that the differences described between lentic and lotic populations of D. ch. chilensis may result from genetic differentiation among populations. This study is an initial contribution to the knowledge of cytogenetic characteristics in Chilean populations of Diplodon chilensis chilensis.