The presence of Anaplasma phagocytophilum, a tick-transmitted zoonotic pathogen, has been reported in Italy in humans and several animal species, including dogs, but data concerning its prevalence in the Italian canine population are lacking. The aim of this study was to determine the prevalence of A. phagocytophilum infection in hunting dogs which live in geographic areas of central Italy where the infection had been previously detected in wild animals. Sera from 215 hunting dogs were tested by indirect immunofluorescence antibody test (IFAT) to detect antibodies to A. phagocytophilum. Buffy coat samples from the same 215 animals were submitted to a polymerase chain reaction (PCR) assay specific for the A. phagocytophilum 16S rRNA gene. Thirty-two (14.8%) dogs proved to be seropositive with antibody titres from 1:40-1:2,560. The highest seroprevalence was observed in dogs 6-10-year-old. Two seropositive (0.9%) animals were also PCR positive. Sequencing of PCR products revealed gene sequences of A. phagocytophilum in both cases. These results confirm the presence of A. phagocytophilum in the Italian areas studied; thus, the zoonotic potential of this agent should be considered particularly for people, as hunters, at risk of exposure to tick bites.