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Cattle are susceptible to infection with multiple serovars of pathogenic leptospires, resulting in abortion, stillbirth, premature birth, reproductive failure and milk drop syndrome. Cattle also act as a reservoir host for <i>L. borgpetersenii</i> serovar Hardjo which is excreted from renal tubules via urine into the environment where it persists in suitable moist conditions. Our previous work demonstrated that 7% of urine samples from beef cattle were positive for <i>L. borgpetersenii</i> serovar Hardjo by culture and/or the fluorescent antibody test (FAT). In this study, a real-time PCR (rtPCR) assay was applied to determine the relative performance of rtPCR based detection of <i>L. borgpetersenii</i> serovar Hardjo compared to previously reported culture and FAT techniques. Of 42 bovine urine samples positive for leptospires by culture and/or FAT, 60% (25/42) were positive by rtPCR. Of 22 culture-positive samples, 91% (20/22) were rtPCR-positive. Of 32 FAT-positive samples, 50% (16/32) were rtPCR-positive. For 10 samples that were culture-positive but FAT-negative, 90% (9/10) were rtPCR-positive. For 20 samples that were FAT-positive but culture-negative, 25% (5/20) were rtPCR-positive. Collectively, these results indicate that no single assay is optimal, and the use of more than one assay to detect leptospires in urine from naturally infected cattle is recommended.