Background and Objectives: Staphylococcus aureus is an important cause of nosocomial and community-acquired infections in every region of the world. Clindamycin is one of the alternative agents used to treat S. aureus infections and accurate identification of clindamycin resistance is important to prevent therapeutic failure. Unfortunately, inducible clindamycin resistance is not detected by standard susceptibility tests. This study aimed to determine the prevalence of the macrolides-lincosamides-streptogramins B (MLSB) resistance in S. aureus isolated in four university hospitals in Tehran, Iran. Material & Methods: Two hundreds and forty-four non-duplicate clinical isolates of S. aureus (133 methicillin resistant S. aureus (MRSA) and 111 methicillin susceptible (MSSA) S. aureus) were collected in 2008. Antimicrobial susceptibilities were determined by the D-test. Results: Altogether, 68% and 61.1% of isolates were resistant to erythromycin and clindamycin, respectively; with higher resistance in MRSA isolates compared to MSSA isolates. The constitutive MLSB (cMLSB) resistance phenotype was recognized in 61.1%, while 5.3% had shown inducible MLSB (iMLSB) resistance phenotype. Constitutive MLSB resistance phenotype predominated over inducible MLSB resistance phenotype and susceptible phenotype (83.9, 9.3 and 6.8%, respectively) among the MRSA isolates, whereas susceptible phenotype predominated over constitutive MLSB resistance phenotype and inducible MLSB resistance phenotype (62.6, 31.3 and 2%, respectively) among the MSSA isolates. Conclusion:Considering the higher prevalence of clindamycin resistance in MRSA isolates compared MSSA isolates, routine D-test of MRSA isolates is strongly recommended to prevent treatment failure.