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Aims To obtain and investigate the potential mechanism for GA3 production in Fusarium fujikuroi GA‐251, a high GA3 producer. Methods and Results Fusarium fujikuroi IMI 58289 was bred with Cobalt‐60 (60Co) radiation and lithium chloride treatment. The best mutant strain GA‐251 was obtained for the subsequent optimization of fermentation conditions. The yield of GA3 by GA‐251 was 2100 mg l−1, while the wild‐type strain was 100 mg l−1, which is a 21‐fold increase in the yield. To elucidate the mechanism of high GA3 yield of GA‐251, the genome was sequenced and compared with wild‐type strain IMI 58289. The results showed 2295 single nucleotide polymorphisms, 1242 small indels and 30 structural variants. These mutations were analysed and enriched in the MAPK signalling pathway, the mRNA surveillance pathway and endocytosis. The potential reasons for the improved GA3 biosynthesis were investigated. Conclusions The potential mechanism of high GA3 yield was attributed to endocytosis pathway and histone modification proteins family. Significance and Impact of the Study A mutant strain GA‐251 in this work that could potentially be utilized in the industrial yield of GA3. The comparative genome analysis would shed light onto the mechanism of yield improvement and be a theoretical guide for further metabolic engineering.