In this study, we identified prognostic biomarkers that predict treatment outcome in patients receiving fertility-preserving high-dose medroxyprogesterone acetate (MPA) therapy through comprehensive multigene panel testing. A total of 38 patients (20 atypical endometrial hyperplasia and 18 stage IA G1 without myometrial invasion) who received first-line MPA therapy were enrolled. Genomic DNA was extracted from formalin-fixed paraffin-embedded samples, and PleSSision-Rapid multigene panel testing was performed. Of the 38 patients, 31 (82%) achieved complete response (CR), 2 (5%) had stable disease (SD), and 5 (13%) had progressive disease (PD) following initial treatment. The median duration to achieve tumor disappearance was 7 months (range: 4-14 months). Following initial treatment, 25 of 32 patients (78%) experienced recurrence, with a median recurrence-free survival (RFS) of 21 months (range: 2-84 months). The most frequently observed actionable gene mutations were PTEN (68.4%), CTNNB1 (55.2%), and PIK3CA (33.3%). Patients harboring PTEN mutations in EMG1 required a significantly longer duration to achieve tumor disappearance (p = 0.011). In addition, the presence of PIK3CA mutations in AEH was significantly associated with shorter RFS (p = 0.048). Molecular classification identified 34 patients (89%) with no specific molecular profile (NSMP), 1 patient (3%) with POLE mutation, and 3 patients (8%) with deficient mismatch repair (d-MMR). Most patients undergoing MPA therapy were classified as having NSMP. Genetic alterations, specifically mutations in PTEN and PIK3CA, were significantly associated with treatment outcomes, highlighting their potential as prognostic biomarkers.