The aim of the present longitudinal field study was to compare the efficiency of 2 analytical approaches for the sanitation of Staphylococcus aureus genotype B (GTB)-positive dairy herds. S. aureus is one of the most widespread mastitis pathogens worldwide. Typically, it causes subclinical, chronic mastitis leading to reduced quality and production of milk, and to substantial economic loss in the dairy industry. In Switzerland, different genotypes of S. aureus have been identified, whereby the genotype B was demonstrated to be the only contagious subtype, causing herd problems. Furthermore, this pathogen can cause food poisoning because of enterotoxin production. As the efficacy of antibiotic therapy and vaccination against S. aureus is not satisfactory, the most promising strategy for controlling this udder pathogen is the implementation of specific sanitation programs. In the present study, a new qPCR assay (very sensitive and specific for S. aureus GTB) was evaluated in the field for the sanitation of GTB-positive herds and compared with classical bacteriology. Both analytical methods were demonstrated to be effective, although the qPCR approach showed some key advantages, which enable the sanitation of entire herds in short time. The use of clean, instead of aseptically collected, milk samples facilitates sample collection in terms of time and cost, enabling the sampling of even big herds during a normal milking time. Because of the high sensitivity of qPCR, the rate of false-negative results is minimal, so that each GTB-positive cow can be correctly identified at any time point during lactation. The conclusive identification of GTB-positive cows can be accomplished within 2 d after sampling: This allows farmers to immediately build milking groups and to maintain the correct milking order. Milk sample analysis becomes easier, faster, more objective, and suitable for routine application. Additionally, all steps of the analytical procedure are suitable for automation, from sample preparation to the final qPCR reaction. This allows for the first time the implementation of sanitation programs at a broader, regional level, instead of being limited to the herd level.