BACKGROUND Ticks and tick-borne diseases are increasingly recognized as a cause of disease in dogs worldwide. The epidemiology of ticks and tick-transmitted protozoa and bacteria has changed due to the spread of ticks to urban and peri-urban areas and the movement of infected animals, posing new risks for animals and humans. This countrywide study reports information on distribution and prevalence of pathogens in ticks collected from privately-owned dogs in Italy. We analyzed 2681 Ixodidae ticks, collected from 1454 pet dogs from Italy. Specific PCR protocols were used to detect i) Piroplasms of the genera Babesia and Theileria, ii) Gram-negative cocci of the family Anaplasmataceae and iii) Borrelia burgdorferi sensu lato. Sequencing of positive amplicons allowed for species identification. RESULTS Babesia/Theileria spp. DNA was detected in 435 homogeneous tick-pools (Minimum Infection Rate (MIR) = 27.6%; 95% confidence interval (CI) = 25.4-29.8%) with higher prevalence in Ixodes ricinus and Rhipicephalus sanguneus group. The zoonotic B. venatorum was the most prevalent species (MIR = 7.5%; 95% CI = 6.3-9.0%). Anaplasma and Ehrlichia species were detected in 165 tick-pools (MIR = 10.5%; 95% CI = 9.3-11.8%) and specifically, A. phagocytophilum was identified with MIR = 5.1% (95% CI = 4.1-6.3%). Borrelia burgdorferi s.l. and B. afzelii were detected with MIR = 0.4% (95% CI = 0.2-0.8%) and MIR = 0.3% (95% CI 0.1-0.7%) respectively. CONCLUSIONS Zoonotic pathogens B. venatorum and A. phagocytophilum were the most frequently detected in ticks collected from privately-owned dogs which might be used as markers of pathogens presence and distribution.