Simple SummaryThe pond slider (Trachemys scripta) is a turtle species native to Central America. Pond sliders have been commercialized as pets since the 1950s, but often ended up being released or escaping into an environment to which they are allochthonous. Trachemys scripta is presently classified as an invasive alien species in Europe and other countries. The introduction of pond sliders in foreign ecosystems has had a deep environmental and ecological impact. Moreover, freed or escaped captive turtles could be carriers of pathogens, such as Chlamydiaceae. In this study, we report the identification of a Chlamydia spp. in two pond sliders found dead after the hibernation period. Further molecular characterization revealed that this Chlamydia is identical to a strain reported in Poland in 2017, found in both captive and free-living freshwater turtles, and has a close phylogenetic relationship to Chlamydia pneumoniae, a species infectious to humans, and to chlamydial strains found in other reptiles. This first finding evidences the presence of this novel Chlamydia in Italian turtles, but further studies will be necessary to evaluate the prevalence in the local turtles’ population and the strain pathogenicity.Trachemys scripta is a turtle species native to Central America. Since the 1950s, pond sliders have been imported worldwide as companion animals, but have often ended up in foreign ecosystems with great ecological consequences. Moreover, both autochthonous and invasive species of turtles can be carriers of pathogens, including Chlamydiaceae. In the present study, pulmonary tissues collected from four Trachemys scripta were tested with a 23S-targeting real-time PCR (rPCR) specific for the Chlamydiaceae family. The turtles were hosted in a rescue center for wild exotic animals located in northeastern Italy, and were found dead after the hibernation period. Two out of four individuals resulted positive in rPCR for the presence of Chlamydiaceae. Further characterization of this positivity was performed by phylogenetic analysis of the 16S rRNA and outer membrane protein A genes. The phylogenetic tree showed that these chlamydial strains are identical to a novel Chlamydia reported in 2017 in Polish freshwater turtles, and closely related to Chlamydia pneumoniae and to other chlamydial strains found in reptiles. This first finding evidences the presence of this Chlamydia strain in Italian turtles, but further studies will be necessary to confirm the presence and the strain pathogenicity and to evaluate its prevalence in the local turtles’ population.