Background: Mycoplasma pneumoniae is one of the widespread causes of community-acquired pneumonia (CAP). Over recent years, the widespread use of macrolides has led to the emergence of macrolide-resistant M. pneumoniae (MRMP) resulted from mutations at specific positions of domain V of the 23S rRNA gene. Methods: We collected 100 samples of throat swabs from patients with respiratory infections. After extraction of DNA from bacterial cell cultured in PPLO broth media using Roche kit (Germany), the PCR was performed on specific samples of M. pneumoniae using specific primers for 23S rRNA gene. Afterwards, for positive samples, minimum inhibitory concentration (MIC) was determined using the broth microdilution with Clarithromycin. Finally, the PCR product was sequenced to detect mutations related to macrolide resistance in domain V of 23S rRNA. Results: According to the analysis of the sequenced PCR product of M. pneumoniae 23S rRNA gene using Clustalw2 online software, one of the samples were shown to have a mutation at A2431G and G2491A positions. The MIC measurement also revealed that all isolates were sensitive to Clarithromycin, and there was no macrolide resistance to Clarithromycin in all isolates. Conclusions: Sequence analysis of the 23S rRNA gene in M. pneumoniae, revealed no macrolide resistance of M. pneumoniae to Clarithromycin. Thus, the use of these antibiotics should be restricted to prevent the development of macrolide-resistant M. pneumoniae in Iran.