Simple SummaryThe Eurasian otter (Lutra lutra) is a semiaquatic mammal listed as endangered in Italy. We conducted a first post mortem (PM) study of 28 otters, mostly collected between 2009 and 2017, in Italy. Vehicle collision was the most common cause of death and mainly involved young animals. The examined otters appeared to be smaller than individuals from northern Europe, and most of them were in good body condition. However, in some individuals, necropsy exams revealed different signs of disease, such as pleuropneumonia and peritonitis, gastritis, lymphoma, or bacterial infection from conspecific bites. Ticks and endoparasites were rarely detected. Data collected on scene of death helped to identify factors forcing otters to move over the road, suggesting road-kill mitigation measures. Comprehensive, standardized PM investigations through collaborative and coordinated research efforts are essential for otter conservation in Italy.Dead specimens provide valuable data for the conservation of threatened species, allowing investigations of mortality, health conditions, and demographic parameters. The Eurasian otter (Lutra lutra) is a semiaquatic carnivore listed as endangered in Italy. In 2009, we started the first post mortem (PM) study of otters in Italy, through collaborative research between mammal ecologists and veterinary pathologists, using standardized protocols. Twenty-eight otters, mostly collected between 2009 and 2017, were examined. Most otters were males (67%), between 1 and 3 years old (64%), and predominantly in good nutritional condition. Adult males were significantly larger than adult females (p < 0.02), as expected for the species, although both sexes appeared to be smaller than otters examined in Central–northern Europe. The youngest sexually mature female was 3 years old. Road traffic collisions were the major cause of death, especially in young individuals, and mainly occurred in autumn–winter, particularly for females. Investigations of the scene of death contributed to revealing factors forcing otters to travel out of the water and move over the road, suggesting appropriate measures to reduce vehicle collision risk. Other causes of death included blunt chest trauma of uncertain origin, dog and conspecific attacks, or diseases of infectious or non-infectious origin, such as ulcerative gastritis, pleuropneumonia and peritonitis. Other diagnosed diseases included lymphoma. Ecto- and endoparasites were rarely detected, although we report the first documentation of heartworm and Ixodes hexagonus infestation in Italian otters. It is important to continue comprehensive, standardized PM investigations of otters in Italy to define baseline health, biometric and demographic parameters, collect biological samples for comparative analyses, and to reduce road-kill mortality. The present study suggests that the timely collection of carcasses and collaborative and coordinated research efforts are essential for obtaining useful data for the conservation of otters.