This study describes for the first time in Ukraine the results of serological screening for antibodies to Borrelia burgdorferi sensu lato (s.l.) in the population of pet and stray dogs. The study was conducted in the city of Bila Tserkva, Kyiv region, in 2021 and 2022. A total of 351 serum samples were examined, of which 168 were collected from pet dogs and 183 from stray dogs. The study included the use of serological methods, including immunochromatographic analysis, enzyme-linked immunosorbent assay and Line blot is a simplified form of Western Blot. The results of the enzyme-linked immunosorbent assay showed that 14 positive samples from stray dogs had specific antibodies to B. burgdorferi s.l. In addition, 10 questionable serum samples were received, all from stray dogs. Further testing of the questionable samples using a Line blot assay showed that five of them contained antibodies to highly specific surface proteins of B. burgdorferi s.l., in particular to p100, VlsE and p18, confirming the presence of a natural infection. The remaining five samples were negative, indicating nonspecific immunoassay reactions. In contrast, the results of immunochromatographic analysis did not reveal any positive serum samples, which casts doubt on the possibility of using this method as a rapid screening tool for seroprevalence studies and requires further investigation. In summary, the study showed that the seroprevalence of Lyme borreliosis among the studied population of stray dogs remained stable between 2021 and 2022, with rates of 10.2% and 10.7%, respectively, without a statistically significant difference (p=0.9164). In contrast, the studied population of pet dogs was consistently seronegative for B. burgdorferi s.l. in both years. Statistical analysis revealed a significant difference in seroprevalence between the stray and pet dog populations studied (p<0.00001). The stability of the seroprevalence of Lyme borreliosis in the population of stray dogs indicates the constant presence and persistence of this zoonosis in the study region. This emphasizes the need to implement long-term surveillance to better understand the dynamics of the disease and the possibility of using dogs, especially stray dogs, as a «marker» species for predicting the risks associated with the spread of Lyme borreliosis. Continuous surveillance is crucial for the development of evidence-based strategies to combat vector-borne diseases within the framework of the One Health concept. Key words: Lyme borreliosis, seroprevalence, dogs, antibodies, B. burgdorferi sensu lato, Ukraine.