This study was carried out to investigate the prevalence of Babesia bovis and B. bigemina in cattle in Erzurum region by Real Time PCR and to characterize the isolates molecularly. Totally 300 whole blood samples from clinically healthy cattle in different ages, sex and breeds were collected into the tubes with EDTA in 2011-2012. Following DNA extraction, Sybergreen and TaqMan probe based Real Time PCR analyses were carried out with B. bovis and B. bigemina primers amplifying msa-2c and rap-1 gene regions. Out of 300 samples, 28 (9.3%) and 17 (5.7%) were found to be infected with B.bovis and B. bigemina. No mix infection was found in the samples. Four of B. bovis and three of B. bigemina isolates were sequenced with respect to msa-2c and rap-1 gene regions for phylogenetic analyses. According to pairwise comparisons of B. bovis isolates, genetic distances were found as 0.6%, 2.3%, and 7.5%, among each other, some other isolates in Turkey, and in the world, respectively. In the phylogenetic analyses of B.bigemina, genetic differences were detected as 0.3% and 0.5% among each other and the other isolates in the world. In the statistical analyses of B. bovis and B. bigemina positive samples according to age, sex and breed of cattle no statistical significance (p>0.05) was founded according to age and breed while the statistical difference according to sex was found significant for B. bigemina (p<0.05), and not significant for B. bovis (p>0.05). In conclusion, the molecular characterization and the prevalence of B. bovis and B. bigemina in cattle in Erzurum region were determined by Real Time PCR. This is the first report on the molecular characterizations of B. bovis and B. bigemina in Erzurum region.