Background and Aim : Methicillin resistant Staphylococcus aureus (MRSA) and methicillin resistant coagulase-negative staphylococci (MRCONS) are one of the important pathogens of nosocomial infections and has shown a frequent and rapid development of antibiotic resistance.The goal of the present study was to evaluate the aminoglycoside modifying enzymes and tetracycline resistance genes in clinical isolates of staphylococci in Shiraz teaching hospitals.Methods : A total of ۱۱۳ staphylococci isolates were recovered from different clinical samples. Antimicrobial susceptibility of isolates against aminoglycoside and tetracycline antibiotics was tested by the Kirby-Bauer disk diffusion method align with CLSI guidelines. Polymerase chain reaction (PCR) for the presence of tet and AMEs genes were carried out among staphylococci isolates.Results : Of ۱۱۳ Staphylococcus isolates, ۳۴.۵% were MRSA, ۳۲.۷% were methicillin sensitive Staphylococcus aureus (MSSA), ۲۶.۵% were MRCONS and ۶.۲% were methicillin resistant coagulase-negative staphylococci (MSCONS). The resistance rates toward tetracycline antibiotics were: ۴۶% to tetracycline, ۱۲.۴% to doxycycline and ۸% to minocycline. Also, the resistance to aminoglycoside antibiotics were: ۴۳.۴% to tobramycin, ۳۲.۷% to amikacin and ۳۱.۹% to gentamicin. The highest frequency in tetracycline resistant genes were among tetM about ۴۲.۵% isolates, followed by tetK in ۳۴.۵% isolates, tetL and tetO in ۲.۷% and ۰.۹% isolates, respectively. The frequency of detected AME genes were aac (۶’)-Ie/aph (۲”), aph (۳’)-IIIa and ant(۴’)-Ia in ۳۹.۸%, ۳۰.۱% and ۱۶.۸% isolates, respectively.Conclusion : Our results showed a high rates of resistance to aminoglycoside and tetracycline antibiotics among MRSA and MRCONS isolates from hospitals in Shiraz. The aac (۶′)/aph (۲′′) and aph (۳’)-IIIa genes were the most identified genes related to aminoglycoside resistance and tetM and tetK genes were the most frequent genes associated with tetracyclines resistance. Therefore implementation of policies in order to prevent arbitrary consumption of antibiotics that would decrease the emergence of resistance to aminoglycoside and tetracycline among MRSA and MRCONS isolates is needed in Iran.