Nuclear or mitocondrial DNA-based genetic markers of invertebrates are used in population genetic as polymorphism markers between individual, species and/or populations. Perumytilus purpuratus is specie widely distributed from Ecuador to Cabo de Hornos, with high fecundity rates and density along the Chilean coasts. With the goal to determine differences in the genetic structure of P. purpuratus populations in polluted areas, we amplified the internal transcribed spacer (ITS) region of the ribosomal DNAfrom Perumytilus purpuratus and we analyzed the restriction fragments polymorphism. The results showed that the P. purpuratus have two ITS-alleles, 3 genotypes and 3 haplotypes (by digestion with TaqaI y HhaI enzymes) which were used to estimate intra-population variability in contaminated or no contaminated areas. Besides, the data indicated a decreasing in genotypes and haplotypes-ITS frequencies values, as well as heterozygote deficiencies and a tendency to disappearance of one allele from P. purpuratus population inhabiting the area with strong influx of domestic and industrial pollution. Such results suggest that ITS can be used as nuclear marker for the genetic analysis of population such as P. purpuratus that probably are changing its genetic structure by environmental stress, and fixing changes on its genotypes and haplotypes frequencies between the populations. Such variation could to maintain the equilibrium in accordance with the environmental conditions uniformity.