Bovine genital leptospirosis (BGL) is a chronic reproductive disease in cattle, often causing significant economic losses, and is commonly associated with leptospiral strains belonging to the Sejroe serogroup. A two-step protocol was recommended, based on serological screening of the herds followed by an individual diagnosis with PCR of a genital sample. Although proposed, it has not been commonly applied under field conditions, leading to frustrating outcomes in disease control. In that context, the present study aimed to demonstrate the viability of that two-step protocol under field conditions for diagnosing BGL in eight herds with reproductive disorders. Blood samples were collected from 440 cows for serology. In addition, 304 cervicovaginal mucus (CVM) samples were collected for