The aim of this investigation was the comprehensive characterization of the in vitro antibiotic resistance profile and of the detectable resistance determinants in a systematic sampling of Staphylococcus aureus isolates from raw milk from clinically healthy cows. Quarter milk samples from all asymptomatic, milk producing cows from 34 Thuringian dairy herds were taken twice (n = 10421 and 10 417 respectively) and investigated bacteriologically. 189 out of the 1902 detected Staphylococcus aureus isolates were selected and established in terms of their resistance pattern using DNA-microarray analysis and agar diffusion test (AVID 1998) for the determination of the genotypic and phenotypic resistance, respectively. 135 isolates (71.4%) did not carry any resistance gene represented on the array. The penicillin resistance gene blaZ was found in 21 isolates (11.1%), which were tested resistant to penicillin by agar diffusion test. Only four isolates (2.1%) harboured the methicillin resistance gene mecA and were thus defined as methicillin-resistant Staphylococcus aureus (MRSA). They belonged to the clonal complex CC398 and possessed, beside mecA, also tetracycline resistance genes. Other resistance markers were very rare, with macrolide and aminoglycoside resistance genes being found in only one isolate (0.5%) each. On one hand, the present survey shows the (still) limited antimicrobial resistance of Staphylococcus aureus isolated from clinically healthy dairy cows. On the other hand, the study demonstrates that MRSA can be detected in raw milk in small quantities, but these strains show only few additional resistance properties.