Babesiosis in dogs is a common tickborne disease in Croatia transmitted by Dermacentor reticulates. Until now, the diagnosis of canine babesiosis has been based on the compatible clinical signs, light microscopy of thin blood smears and positive reaction to antibabesial treatment, which are not recommended methods for giving the definitive diagnosis of babesia infection. The aim of the present study was to detect and characterize the species and subspecies of Babesia spp. that cause canine babesiosis in Croatia using PCR and sequencing analysis. A hundred dogs with typical signs of babesiosis, who came to the Clinic for Internal Diseases in Zagreb during 2004 and 2005 were included in this study. The blood smears from 86 of the dogs showed the presence of large (4- 5µ ; ; m) single or paired intraerythrocytic, pyriform trophozoites of Babesia canis. Among these dogs 28 were randomly chosen for further study. The most common clinical and hematological signs were lethargy, anorexia, fever, dark urine and thrombocytopenia. The results of this study showed the detection of one subspecies, namely Babesia canis canis using PCR and subsequent sequence analysis of portions of the nns rRNA gene in 27 of 28 samples. The sequencing analysis of the studied isolates showed 100% homology in eleven samples, 99.7% homology in eleven samples (one nucleotide difference) and 99.4% homology in five samples (two nucleotide difference) with Babesia canis canis. Nucleotide sequence analysis suggested that Babesia canis canis could be divided to three genetically different groups.