Population size of European otters (Lutra lutra) was estimated in Pollino National Park (southern Italy) by genetic typing of fresh feces collected in the field. Of 187 fecal samples gathered, 185 (98.9%) yielded otter DNA, 77 (41.2%) were successfully typed, and 23 different genotypes were identified. A nonlinear regression between the number of typed spraints and the cumulative number of identified genotypes was repeated after randomization of the sample until it gave an estimated otter population of 34–37 animals (0.18–0.20 otters/km of watercourse). The applied method represents a valuable conservation tool, combining the advantages of an indirect survey with the accuracy of an exhaustive census.