This study was carried out to compare Reverse Line Blotting (RLB), Nested PCR and Real Time PCR techniques in the molecular diagnosis of Babesia bovis and Babesia bigemina in cattle. Genomic DNA extractions were performed on 400 blood samples which were previously collected from cattle in various provinces of Turkey and stored in the laboratory with respect to use in different project studies. The concentrations of the DNAs were measured in NanoDrop spectrophotometer and analyzed by RLB, Nested PCR and Real Time PCR techniques after preparing the suitable concentrations. Totally 18 (4.50%), 59 (14.75%), 16 (4.00%) and 2 (0.50%) of examined samples were found to be infected with B. bovis, B. bigemina, Babesia spp. and B. bovis + B. bigemina mix, respectively by RLB. 23 (5.75%), 71 (17.75%), 7 (1.75%) and 23 (5.75%), 75 (18.75%), 9 (2.00%) of the examined samples were found to be infected with B. bovis, B. bigemina and B. bovis + B. bigemina mix by Nested PCR and Real Time PCR, respectively. When comparing the Nested PCR and RLB results with Real Time PCR assay, 94.4% and 88.8% sensitivity and both 100.0% specificity were determined, respectively. 5, 9 and 2 out of the total 16 Babesia spp. positivity's in RLB test were determined as B. bigemina, B. bovis, B. bovis + B. bigemina mix, respectively by both Real Time and Nested PCR. In conclusion, Real Time PCR was found to be more sensitive than Nested PCR and RLB in the investigation of B. bovis and B. bigemina in cattle with this study.