Canine babesiosis is an emerging tick-borne disease of major veterinary concern in Europe. Its prevalence has increased in the last two decades and is spreading rapidly toward the north. The aim of this study was to investigate the genetic diversity ofBabesiaspp. strains isolated from naturally infected dogs in a tick-endemic area (Dobrogea) in southeastern Romania. For this purpose, a total of twenty-three samples from dogs diagnosed with various clinical forms of babesiosis, evaluated by means of clinical history, physical examination, and hematological tests, were subjected to a molecular investigation using PCR, sequencing analysis, and genetic characterization. A microscopic examination of thin Diff-quick-stained blood smears revealed large intra-erythrocyticBabesiapiroplasms in all dogs. The PCR and sequencing analysis results indicated the presence ofBabesia canisin 22 dogs (95.7%) andBabesia vogeliin 1 dog (4.3%). Among theB. canisisolates, two genotypes were distinguished based on two nucleotide substitutions (GA→AG) observed in the 18S rRNA gene sequences (at positions 609 and 610), with the AG genotype predominating (54.5% of samples), while the GA variant was identified in 9.1% of samples. In the remaining isolates (36.4%), both variants were identified. TheB. vogeli-positive dog also tested positive for antibodies againstEhrlichia canisand displayed severe disease. This study reports, for the first time, the presence of genetically heterogenicB. canisstrains in dogs with clinical babesiosis in Romania. These findings provide a basis for future studies on the relationship between the genetic structure of the causative agents of canine babesiosis in Romania and the course of the disease.