Objective To analyze the clinical data of a rare case of gastrointestinal stromal tumors （GISTs） with multifocal heterochronous primary resistance due to temporal gene mutations after long-term treatment with imatinib， and to reveal the importance of secondary mutation heterogeneity in the recurrence and resistance process of GISTs. Methods The clinical data of one patient with recurrent GISTs were collected. The histomorphology observation was observed by HE staining；the expressions of related proteins were detected by immunohistochemistry；first and second generation sequencing techniques were used to analyze the tumor gene mutations. Results The patient， a 66-year-old male， underwent partial gastrectomy for gastric stromal tumor.The first-generation sequencing results identified it was a proto-oncogenea and there was a deletion mutation in exon 11 of the tyrosine protein kinase kit （KIT） gene （p.551-554del）. Approximately one year after imatinib treatment and a subsequent 4-month drug holiday， the patient experienced a relapse with a splenic lesion.The continuous first-line treatment with imatinib controlled the splenic lesion. After 59 months， a new pelvic mass was identified， and the patient received increased doses of imatinib （600 mg·day^－1） along with second-line therapy with sunitinib. After treated for two months，the CT scan results showed that the size of the splenic lesion tended to stabilize， while the volume of the new pelvic lesion continued to increase. The second generation sequencing results indicated that on the basis of the original KIT gene exon 11 deletion， subsequent mutations in exon 13 （p.V654A） and exon 17 （p.Y823D） of the KIT gene occurred separately in two recurrent lesions. Conclusion Under the pressure of targeted drug selection， the biological behavior of GISTs shows complexity，such as，time heterogeneity，spatial heterogeneity and molecular heterogeneity； different recurrent lesions may exhibit different resistance mechanisms.