Background: The aim of this study was to determine exposure to vector-borne pathogens (VBPs) in populations of dogs living on Greek islands in the Ionian and Aegean seas. Methods: In total, 1154 dogs with different lifestyles and of varying ages and breeds were randomly sampled and examined for the presence of clinical signs compatible with canine vector-borne diseases (CVBDs). Blood was collected from each individual animal. For the detection of antibodies against Leishmania spp., the WITNESS ® Leishmania test was performed, and positive samples were further examined with indirect enzymatic immunoassay (ELISA). Antibodies to Borrelia burgdorferi, Ehrlichia canis or E. ewingii, as well as Anaplasma phagocytophilum or A. platys were investigated using the Snap ® 4Dx ® Plus test. Positive Ehrlichia spp. and Anaplasma spp. samples were further examined using an indirect ELISA for further identification of the species. Results: In total, 25.6% of dogs were exposed to at least one of the pathogens investigated, with seroprevalences varying regionally. Of these seropositive dogs, 27.4% displayed clinical signs suggestive of CVBDs, such as cutaneous lesions, enlarged lymph nodes, pale mucous membranes, onychogryphosis and weight loss. The overall seroprevalence detected using the rapid tests was 15.3% for Leishmania spp., whereas 2.3% of the examined dogs were found to be positive for Anaplasma spp. and 7.5% for Ehrlichia spp. while B. burgdorferi was not detected. Twenty-four samples positive to A. phagocytophilum by ELISA were analysed by PCR for the presence of Anaplasma DNA. PCR and sequencing results showed the presence of A. platys DNA in 4 samples and E. canis DNA in 4 samples. The remaining samples (66.7%) were negative. Conclusions: In the present study, exposure of dogs to VBPs was shown in the geographical areas investigated. Results confirm that on Greek islands VBPs represent a constant health risk for both native and visiting dogs, suggesting the presence of distinct "hot-spots" of VBP infections on different islands. In order to reduce the risk of transmission and the spread to non-endemic regions, the protection of dogs through use of repellents and vaccines, together with owner education, seem to be of paramount importance.