Background: Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a significant pathogen in community and hospital environments and is associated with high mortality and morbidity. Both polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) methods are sensitive and acceptable molecular methods for the diagnosis of infectious diseases. Objectives: This study aimed to develop detection assays for Staphylococcal mecA and spa using multiplex PCR and LAMP. Methods: Both methods were standardized, and detection limits were determined using serial dilutions of S. aureus DNA samples. Fifty-three clinical isolates of S. aureus were confirmed to the species level using biochemical tests and multiplex PCR and multiplex LAMP for the spa gene, while disk diffusion, minimum inhibitory concentration, and detection of mecA genes were used for the assessment of methicillin resistance. Results: The PCR could detect the mecA and spa genes at 1 fg/mL and 10 fg/mL of bacterial DNA, which were equal to 35 and 350 gene copy numbers, respectively. Similarly, multiplex LAMP detected the spa and mecA genes at 0. 1 fg/mL and 1 fg/mL of bacterial DNA, which were equal to 3. 5 and 35 genome copy numbers, respectively. According to MIC and disk diffusion methods, four (7. 54%) cases were oxacillin-sensitive methicillin-resistant S. aureus, 16 isolates were methicillin-sensitive, and 37 isolates were methicillinresistant. According to multiplex PCR, 47. 75% of the isolates were mecA-positive while in multiplex LAMP, 41 (35. 77%) isolates were mecA-positive. Conclusions: The sensitivity and specificity of the multiplex LAMP were higher than those of multiplex PCR and biochemical methods. Thus, we can apply the LAMP for the routine detection of MRSA.