Background: Coagulase Negative staphylococci (CoNS) are nosocomial pathogens. The main objective of the study was to compare the performance of disk diffusion, E-test and agar screening methods with Real-time PCR technique for detection of methicillin resistance in coagulase negative staphylococci (MRCoNS), using Taqman® Real-time PCR for mecA as the “gold standard” comparison assay. Methods: 88 coagulase negative Staphylococcus isolates were identified out of 284 Staphylococcus isolates collected from Hajar and Kashani hospitals-shahrekod, Iran. Methicillin resistance strains were identified by several methods: Disk diffusion, Agar screening, E-test and Real-time PCR. The results of the tested methods were compared with those of the Real-time PCR by Chi square or Fisher’s exact tests. Results: Of the 88 coagulase negative Staphylococcus isolates tested, 46 isolates (52.3%) were mecA-positive and 42 isolates (47.7%) were mecA-negative. The results of all the tested methods had a statistically significant agreement with those of Real-time PCR. The E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen (6.0 μg/ml) method were 96% and 98%, respectively and the sensitivity and specificity of oxacillin Disk diffusion method were 91% and 90%, respectively. Conclusion: In the present study, E-test is proposed as the best phenotypic method. In case economic issue matters, the oxacillin agar screening method (6.0 μg/ml), which is suitable for the detection of MRCoNS due to its accuracy and low cost, is recommended.