BackgroundMycoplasma pneumoniae (M. pneumoniae) is an important pathogen of community-acquired pneumonia in children. The factors contributing to the severity of illness caused by M. pneumoniae infection are still under investigation. We aimed to evaluate the sensitivity of common M. pneumoniae detection methods, as well as to analyze the clinical manifestations, genotypes, macrolide resistance, respiratory microenvironment, and their relationship with the severity of illness in children with M. pneumoniae pneumonia in Wuhan.ResultsAmong 1,259 clinical samples, 461 samples were positive for M. pneumoniae via quantitative polymerase chain reaction (qPCR). Furthermore, we found that while serological testing is not highly sensitive in detecting M. pneumoniae infection, but it may serve as an indicator for predicting severe cases. We successfully identified the adhesin P1 (P1) genotypes of 127 samples based on metagenomic and Sanger sequencing, with P1-type 1 (113/127, 88.98%) being the dominant genotype. No significant difference in pathogenicity was observed among different genotypes. The macrolide resistance rate of M. pneumoniae isolates was 96% (48/50) and all mutations were A2063G in domain V of 23S rRNA gene. There was no significant difference between the upper respiratory microbiome of patients with mild and severe symptoms.ConclusionsDuring the period of this study, the main circulating M. pneumoniae was P1-type 1, with a resistance rate of 96%. Key findings include the efficacy of qPCR in detecting M. pneumoniae, the potential of IgM titers exceeding 1:160 as indicators for illness severity, and the lack of a direct correlation between disease severity and genotypic characteristics or respiratory microenvironment. This study is the first to characterize the epidemic and genomic features of M. pneumoniae in Wuhan after the COVID-19 outbreak in 2020, which provides a scientific data basis for monitoring and infection prevention and control of M. pneumoniae in the post-pandemic era.