Background <p>To analyze the clinical characteristics of Mycoplasma pneumoniae pneumonia in children with 23&#xa0;S rRNA mutations in Domain V and to explore the optimal timing of glucocorticoid therapy when fever persists despite macrolide antibiotic treatment.</p> Methods <p>This study retrospectively analyzed clinical data from 350 children hospitalized with Mycoplasma pneumoniae pneumonia (MPP) between November 2022 and October 2023. Patients were stratified into a mutation-positive group (<i>n</i> = 286) and a mutation-negative group (<i>n</i> = 64) based on the presence of 23&#xa0;S rRNA Domain V mutations in Mycoplasma pneumoniae. The clinical characteristics, laboratory results and imaging manifestations of the two groups were compared first. In this retrospective study, children with mutation-positive MPP treated with azithromycin were further grouped by persistent fever duration at the time of glucocorticoid initiation: Group A (<i>n</i> = 60, 3 days), Group B (<i>n</i> = 63, 5 days), and Group C (<i>n</i> = 64, 7 days). All received glucocorticoids after the respective fever milestones, with outcomes compared across groups. Furthermore, Receiver Operating Characteristic (ROC) curves were constructed using laboratory indicators to identify predictive markers for severe MPP (SMPP).</p> Results <p>There were statistically significant differences between the 23&#xa0;S rRNA Domain V mutation-positive and negative groups in 24/48/72-hour defervescence rates, pulmonary consolidation rate, white blood cell count, neutrophil count, high-sensitivity C-reactive protein (hs-CRP), serum amyloid A (SAA), SAA/hs-CRP ratio, lactate dehydrogenase (LDH), D-dimer, fever duration, and hospital stay (<i>P</i> &lt; 0.05). Both Group A and Group B showed significantly higher 48/72-hour defervescence rates than Group C (<i>P</i> &lt; 0.05), while Group A had shorter fever duration and hospital stay than Groups B and C (<i>P</i> &lt; 0.05). Additionally, Group A had a lower severe pneumonia rate than Group C (<i>P</i> &lt; 0.05), which suggested that early glucocorticoid therapy may be associated with potentially improved clinical outcomes. ROC curve analysis demonstrated that hs-CRP, LDH, D-dimer, and their combined detection had high predictive value for SMPP.</p> Conclusions <p>The 23&#xa0;S rRNA Domain V mutation shows significant association with clinical characteristics of pediatric MPP. Timely glucocorticoid intervention can substantially improve patient outcomes. Furthermore, combined measurement of hs-CRP, LDH, and D-dimer demonstrates significant predictive value for early identification of SMPP.</p> Clinical trial number <p>Not applicable. This retrospective analysis was conducted using existing data and did not involve direct intervention with participants.</p>