This study investigates the molecular epidemiology of Mycoplasma pneumoniae (M. pneumoniae) pneumonia in children in Wuhan, China, from 2020 to 2022. A total of 1,259 clinical samples were analyzed, with 461 positive for M. pneumoniae via quantitative PCR (qPCR). Serological testing showed lower sensitivity but could predict severe cases. The adhesin P1 genotype was identified in 127 samples, with P1-type 1 being dominant (88.98%). No significant difference in pathogenicity was observed among genotypes. Macrolide resistance was high (96%), with mutations in the 23S rRNA gene. No significant difference in upper respiratory microbiome was found between mild and severe cases. The main circulating M. pneumoniae was P1-type 1, with a high macrolide resistance rate. qPCR was effective for detection, and IgM titers exceeding 1:160 may indicate severe illness. No direct correlation was found between disease severity and genotypes or respiratory microbiome. This study provides insights into M. pneumoniae epidemiology and genomics in Wuhan post-COVID-19, supporting monitoring and infection control. Key findings include the efficacy of qPCR, the potential of IgM titers as severity indicators, and the lack of direct correlation between severity and genotypic or microbiome factors. The study highlights the importance of monitoring M. pneumoniae genotypes and resistance, especially with the rise of macrolide-resistant strains. It also emphasizes the need for improved diagnostic methods and surveillance systems to better manage M. pneumoniae infections.This study investigates the molecular epidemiology of Mycoplasma pneumoniae (M. pneumoniae) pneumonia in children in Wuhan, China, from 2020 to 2022. A total of 1,259 clinical samples were analyzed, with 461 positive for M. pneumoniae via quantitative PCR (qPCR). Serological testing showed lower sensitivity but could predict severe cases. The adhesin P1 genotype was identified in 127 samples, with P1-type 1 being dominant (88.98%). No significant difference in pathogenicity was observed among genotypes. Macrolide resistance was high (96%), with mutations in the 23S rRNA gene. No significant difference in upper respiratory microbiome was found between mild and severe cases. The main circulating M. pneumoniae was P1-type 1, with a high macrolide resistance rate. qPCR was effective for detection, and IgM titers exceeding 1:160 may indicate severe illness. No direct correlation was found between disease severity and genotypes or respiratory microbiome. This study provides insights into M. pneumoniae epidemiology and genomics in Wuhan post-COVID-19, supporting monitoring and infection control. Key findings include the efficacy of qPCR, the potential of IgM titers as severity indicators, and the lack of direct correlation between severity and genotypic or microbiome factors. The study highlights the importance of monitoring M. pneumoniae genotypes and resistance, especially with the rise of macrolide-resistant strains. It also emphasizes the need for improved diagnostic methods and surveillance systems to better manage M. pneumoniae infections.