Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, which is often empirically treated with macrolides or azalides such as erythromycin or azithromycin. Recent studies have discovered the existence of macrolide-resistant strains within the population that have been mapped to mutations within the domain V region of the 23S rRNA gene. Currently, identification of these resistant strains relies on timeconsuming and labor-intensive procedures such as restriction fragment length polymorphism, MIC studies, and sequence analysis. The current study reports two distinct real-time PCR assays that can detect the A2063G or A2064G base mutation (A2058G or A2059G by Escherichia coli numbering) conferring macrolide resistance. By subjecting the amplicon of the targeted domain V region of the 23S rRNA gene to a high-resolution melt curve analysis, macrolide-resistant strains can quickly be separated from susceptible strains. Utilizing this method, we screened 100 clinical isolates and found 5 strains to possess mutations conferring resistance. These findings were concordant with both sequencing and MIC data. This procedure was also used successfully to identify both susceptible and resistant genotypes in 23 patient specimens. These patient specimens tested positive for the presence of M. pneumoniae by a separate real-time PCR assay, although the bacteria could not be isolated by culture. This is the first report of a real-time PCR assay capable of detecting the dominant mutations that confer macrolide resistance on M. pneumoniae, and these assays may have utility in detecting resistant strains of other infectious agents. These assays may also allow for clinicians to select appropriate treatment options more rapidly and may provide a convenient method to conduct surveillance for genetic mutations conferring antibiotic resistance.Mycoplasma pneumoniae is a human respiratory pathogen that is responsible for approximately 20% of all communityacquired pneumonia (14,36,39). Transmission among household contacts typically precedes the recognition of a community-wide outbreak, and the majority of exposed individuals may be asymptomatic or have mild illness and not seek care (2, 8). Nonetheless, severe disease involving multiple organs can occur and can lead to encephalitis and/or death (1,5,6,32,38). Recent outbreaks of M. pneumoniae infections have established that this agent is capable of causing a significant amount of morbidity and occasional mortality in the community (1,18,38). Previous studies have demonstrated that in outbreaks associated with closed settings, such as schools and hospitals, implementing appropriate antimicrobial prophylaxis treatment in a timely manner may shorten the course of the outbreak (12, 13). Because M. pneumoniae lacks a cell wall, infection is treated primarily with macrolide or azalide antibiotics such as erythromycin or azithromycin, although doxycycline and levofloxacin have also been used (4, 12, 13, 25). However, resistance to the macrolides and related azalides has been...