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Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), such as clonal-complex (CC)398, are of clinical relevance due to their multi-drug resistance profiles, adding to the overall burden of MRSA in humans. The objective was to evaluate the double-zone hemolysis (DZH) test as a simple and reliable method for detecting LA-MRSA in the clinical microbiology laboratory. S. aureus isolates assigned to CC398 ( n = 183; 152 MRSA/31 methicillin-susceptible S. aureus [MSSA]), CC1 ( n = 44; MRSA), and other CCs ( n = 144; 94 MRSA/50 MSSA) were investigated. These isolates were screened for DZH on sheep blood agar plates after incubation at 37°C for 24 h. Identification of the scn (human adaptation marker) and hlb genes (encoding hemolysin, intact or truncated) was performed by PCR. The positive and negative predictive values (PPV and NPV), sensitivity (SS), and specificity (SP) of the DZH test were determined. The DZH-positive phenotype was observed in 94.7%, 25%, and 6.4% of MRSA-CC398, MRSA-CC1, and MRSA of other lineages, respectively. Moreover, the DZH-positive phenotype was identified in 9.7% of MSSA-CC398 isolates but not in other MSSA lineages. All 164 DZH-positive isolates carried hlb intact, and 99.4% was scn negative, suggesting an animal origin. Of the 207 DZH-negative isolates, 99.5% was scn positive (indicating human adaptation), and 95.2% possessed a truncated hlb gene. The PPV/NPV/SS/SP values (in %) of the DZH test were as follows: detection of (i) LA-MRSA-CC398: (87.8/96.1/94.7/90.9); (ii) LA-MRSA-CC398/CC1 scn negative: (94.5/100/100/95.8); and (iii) S. aureus scn negative: (99.4/99.5/99.4/99.5). The DZH is a reliable strategy to detect and distinguish LA-MRSA in the clinical microbiology laboratory and is recommended as an adjunct diagnostic test., Importance: This study evaluated a simple and reliable phenotypic test that can be very useful in the clinical microbiology laboratory to detect livestock-associated (LA) methicillin-resistant Staphylococcus aureus (MRSA) isolates and S. aureus of potential animal origin. The proposed double-zone hemolysis test has shown high positive and negative predictive values, sensitivity, and specificity to detect these LA-MRSA clones and S. aureus of potential animal origin. Most LA-MRSA clones exhibit resistance to different classes of antibiotics, with unique epidemiological characteristics, and their early detection has public health relevance and patient management.