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The present study aimed to determine the prevalence of zoonotic vector-borne pathogens, including <i>Anaplasma platys</i>, <i>Anaplasma phagocytophilum</i>, <i>Borrelia burgdorferi</i> sensu lato, <i>Ehrlichia canis</i> and <i>Rickettsia</i> spp. in shelter dogs from Cuba. Blood samples were collected from 100 shelter dogs and examined by molecular methods. Overall, 85 (85%; 95% CI: 77.88–92.12) dogs tested positive for at least one vector-borne pathogen using species-specific qPCR assays. Among the positive samples, <i>E. canis</i> was the most prevalent 62% (95% CI: 52.32–71.68), followed by <i>A. platys</i> 40% (95% CI: 30.23–49.77) and <i>Rickettsia felis</i> 27% (95% CI: 18.15–35.85), whereas 36% (95% CI: 26.43–45.57) showed co-infections. All samples were negative for <i>A. phagocytophilum</i> and <i>B. burgdorferi</i> s.l. The presence of 248 <i>Rhipicephalus sanguineus</i> ticks collected from the dogs was not statistically associated with the occurrence of infections. Thrombocytopenia was the most frequent haematological alteration found in PCR-positive dogs; it was statistically associated with the presence of <i>E. canis</i>, as well as co-infections (<i>p</i> < 0.05). The phylogenetic analyses of <i>A. platys</i> and <i>E. canis</i> based on <i>16S</i> rRNA, <i>groEL</i> and <i>gltA</i> genes showed a low genetic diversity between Cuban strains. The present study demonstrates the high prevalence of vector-borne pathogens with zoonotic potential in shelter dogs from Cuba.