To understand the prevalence and drug resistance of Staphylococcus aureus in different types of milk in Shandong, bacterial isolation, identification and minimal inhibitory concentration (MIC) test were carried out on mastitis and non-mastitis milk samples from 37 farms. The isolation and identification of Staphylococcus aureus were carried by conventional method and PCR technology, the MIC test was conducted with broth micro dilution methods and the data were analyzed by chi-square test. ①A total of 150 strains of Staphylococcus aureus were isolated with the detection rate of 11.26% (150/1332), and the milk samples of mastitis was significantly higher than that of non-mastitis (P=0.009). ②The distribution tend of MIC of 12 drugs was roughly the same as that of EUCAST. ③The isolated strains were the most resistant to penicillin (91.38%), and there was a significant difference in mastitis and non-mastitis milk samples (P=0.047). ④In 116 separation strains, there were 34 (29.31%) strains of multiple resistance (≥3) and there was no significant difference in mastitis and non-mastitis milk (P=0.611). ⑤The resistance rate of six drugs in Weihai was more than 60% and the resistance rate of only one or two drugs in other regions was above 50%. The detection rate of Staphylococcus aureus in mastitis milk samples was significantly higher than that in non-mastitis milk samples. The isolated strain was highly resistant to penicillin and the problem of multiple drug resistance of mastitis milk samples was serious. Different regions showed different levels of drug resistance.