AbstractBackgroundBovine genital leptospirosis (BGL) causes chronic reproductive disease in cattle. This study aimed to apply a combined serological‐molecular testing protocol under field conditions for diagnosing BGL in cows with gestational losses.MethodsThree beef herds with reproductive failures were studied, and 60 cows with gestational losses (20 from each herd) were randomly selected for laboratory diagnosis of BGL. In addition, 40 cows with normal pregnancy were included as a control. Blood samples were collected from all 100 cows for microscopic agglutination testing, and cervicovaginal mucus (CVM) samples were collected from 28 cows with gestational losses and 20 control cows for lipL32‐PCR.ResultsAll herds had high Leptospira seroreactivity (>65%), mainly against serogroup Sejroe. Ten of the 28 CVM samples from cows with gestational losses were PCR‐positive, while all samples from the control group were negative (p < 0.05).LimitationsUnfortunately, the positive samples did not amplify in secY‐PCR for nucleotide sequencing, which would allow the identification of leptospiral strains.ConclusionSerology was sufficient to indicate leptospirosis at the herd level, but the definitive diagnosis of BGL was only possible using CVM PCR. Although seroreactivity against serogroup Sejroe has been associated with gestational losses, this is the first study to conduct CVM PCR as a confirmatory test for BGL diagnosis in extensive beef herds under field conditions.