Influenza D virus (IDV) is a respiratory pathogen affecting the health of cattle, thereby causing economic losses. This study was conducted to detect and isolate IDV, investigate its molecular characteristics, and examine the antibody response to IDV in naturally infected cattle. Real-time RT-PCR was used to analyse 279 nasal swabs and blood sera collected from cattle on farms in the Thrace district of Türkiye, bordering the European Union, for IDV-RNA. One sample from 2020 from a small, family-owned farm (D/OK lineage), two samples from 2021 from an integrated farm (D/OK lineage), and two samples from 2024 (D/Yama2019) from an integrated farm were found to be positive for IDV-RNA. All five samples were collected from farms located in K?rklareli. Positive samples were subjected to virus isolation and phylogenetic analyses. Serum samples were analysed using ELISA and HI tests. Of the 279 samples collected, 1.79% tested positive for IDV-RNA in real-time RT-PCR. Seroprevalence, measured for the first time in Türkiye, was found to be 45.5% and 55.19% by ELISA and HI tests, respectively. Key factors statistically associated with IDV seropositivity included the study year, geographical location, and farm type. Clinically, nasal discharge and cough were the primary symptoms in seropositive animals. Genetic analysis revealed that the 2020–2021 samples clustered within the D/OK lineage, while the 2024 samples belonged to the D/Yama2019 lineage. The findings suggest the co-circulation of multiple IDV strains, although this should be interpreted cautiously given the limited number of sequences in the Thrace region. Considering the lack of an influenza D virus (IDV) vaccine, these findings emphasise the importance of continued surveillance and diagnostic testing of cattle to limit viral spread, protect cattle health, and reduce economic losses associated with IDV.