BackgroundStaphylococcus aureus(S. aureus) causes different types of human infections and can develop resistance to many antibiotics. There is a scarcity of data on themecAgene and multidrug-resistant (MDR) strain distribution of this organism in developing countries, such as Ethiopia. This study investigated the presence ofmecAgene and MDR profile ofS. aureusamong patients attending referral hospitals of Amhara regional state.MethodsOf the total of 110 isolates collected from Amhara regional referral hospitals, 70 MDR isolates were further processed for isolation ofS. aureus mecAgene. Genomic DNA was isolated using a Sigma-Aldrich genomic DNA isolation kit for Gram-positive bacteria. Amplification ofS. aureus mecAgene was performed with the amplicon size of 533 bp. Antimicrobial susceptibility test including methicillin resistance was determined by the Kirby–Bauer disc diffusion method.ResultsThe majority of the isolates were recovered from patients aged less than 5 years (51; 36.7%) and the least number of isolates was recorded in age group greater than 60 years (6; 4.3%). Most of the isolates were from blood (61; 43.9%), followed by wounds (45; 32.4%). A high resistance rate was observed in penicillin (81; 73.6%), followed by cotrimoxazole (78; 70.9%), ceftriaxone (76; 69%), erythromycin (66; 60%), and tetracycline (65; 59.1%). Phenotypically, considering cefoxitin as a surrogate marker, 38 (34.5%) of the isolates were methicillin-resistant. The overall MDR isolates were 80 (72.7%). The PCR amplification result of themecAgene was 14 (20%).Conclusions and Recommendations. High rates of MDR and methicillin-resistantS. aureuswere reported. PCR amplification indicated that 20% of MRSA isolates were themecAgene carriers. Large-scale studies for the detection of MDR strains ofS. aureusincluding MRSA using molecular techniques should be encouraged in the Amhara region.