The canine adenovirus type 2 (CAdV-2) is associated with the infectious tracheobronchitis commonly called “kennel cough”, cosmopolitan in dogs but little explored in gray wolves. Our goals were (i) to evaluate the presence and circulation of CAdV-2 in free-ranging Italian wolves (Canis lupus italicus), through the analysis of spleens and tongues collected from 56 carcasses sampled in three Italian regions between August 2017 and July 2020, and (ii) to support the validity of a matrix such as the tongue, which was never used before. Samples were screened for the presence of CAdV-2 DNA using both PCR and real-time PCR assay. Positive results were related to sampling year, location, sex, age, genetic determination of species, and matrices tested. Three male wolves (5.4%) tested positive in tongue samples, demonstrating that the tongue is an excellent matrix for the detection of CAdV-2. To the best of our knowledge, no studies were performed to evaluate the usability of tongue samples to detect CAdV-2 DNA in grey wolves or other wild animals. The number of wolves tested positive suggests that, during the studied years, the circulation of CAdV-2 in Italian wolves showed a low frequency, consistent with irregular introductions of the virus by dogs or other wild carnivores in these populations. This preliminary study provides new data on the ecology of CAdV-2 in Italian wolves, although future studies are needed to fully understand its real circulation at a national scale, its pathogenetic role in gray wolves, and its risk of transmission to other wild carnivores.