•First molecular report and characterization of Mycoplasma haemocanis and ‘Candidatus mycoplasma haematoparvum’ isolates in dogs (Canis lupus familiaris) from cuba.•Overall, PCR results revealed 15.1% (59/391; 95% CI: 11.5–18.7) positive dogs for Mycoplasma haemocanis, 4.4% (17/391; 95% CI: 2.3–6.4) for ‘Candidatus mycoplasma haematoparvum’, and 1.5% (6/391; 95% CI: 0.3–2.8) showed dual infection.•All tested rhipicephalus sanguineus sensu lato tick samples were PCR-negative, but the occurrence of tick infestation was significantly associated with canine haemoplasma infection in dogs.•DNA sequencing and phylogenetic analysis based on 16S rRNA gene sequences revealed low genetic variability amongst Mycoplasma haemocanis and ‘Candidatus mycoplasma haematoparvum’ isolates from cuba. Haemotrophic mycoplasmas (haemoplasmas) are unculturable, epicellular, cell wall-less gram-negative bacteria distributed worldwide, which infect several mammalian species. In dogs, Mycoplasma haemocanis and ‘Candidatus Mycoplasma haematoparvum’ have been reported as causative agents of infectious anaemia, especially in splenectomised or immunocompromised animals. The present cross-sectional study aims to assess the prevalence, risk factors, and molecular characterization of canine haemoplasmas in Cuba. A total of 391 dog blood samples and 247 tick samples were tested for the presence of canine haemoplasmas by species-specific quantitative TaqMan® real-time PCR assays. Overall, 17.9% (70/391; 95% CI: 14.1–21.7) blood samples were PCR-positive for at least one canine haemoplasmas species, where 15.1% (59/391; 95% CI: 11.5–18.7) for Mycoplasma haemocanis, 4.4% (17/391; 95% CI: 2.3–6.4) for ‘Candidatus Mycoplasma haematoparvum’, and 1.5% (6/391; 95% CI: 0.3–2.8) were co-infected. All collected ticks were identified morphologically as Rhipicephalus sanguineus sensu lato, and none of the tested tick samples was found PCR-positive for the presence of Mycoplasma haemocanis and ‘Candidatus Mycoplasma haematoparvum’. Risk factors for canine haemoplasmas species infection included the presence of tick infestation, crossbreeding and living in kennels, while no association was found with the occurrence of anaemia. Phylogenetic analyses based on the 16S rRNA gene sequences of Mycoplasma haemocanis and ‘Candidatus Mycoplasma haematoparvum’ revealed >99% identity to other isolates distributed worldwide, indicating low genetic variability amongst these canine haemoplasmas species. To the best of the authors´ knowledge, this is the first molecular evidence of Mycoplasma haemocanis and ‘Candidatus Mycoplasma haematoparvum’ infections in dogs from Cuba.