The aim of this study was to investigate the associations between secondary mutations of c-KIT/PDGFRα and acquired imatinib resistance or efficacy of sunitinib in Chinese patients with gastrointestinal stromal tumors (GISTs). Mutations of c-KIT (exons 9, 11, 13, 14, 17, and 18) and PDGFRα (exons 12 and 18) in tumor samples of 50 patients were analyzed by direct sequencing. A total of 50 samples before imatinib and 52 samples after imatinib were collected. Among 52 samples after imatinib, 38 samples were imatinib resistant and 14 samples were imatinib sensitive. All patients before imatinib treatment had primary mutations of c-KIT exon 11 (n = 45) or exon 9 (n = 5), and no PDGFRα mutations were found in these patients. After imatinib treatment, 25 of 38 (65.8 %) resistant tumors had secondary mutations in c-KIT exon 13 (n = 10), exon 14 (n = 1), exon 17 (n = 12) and exon 18 (n = 2), while no secondary mutations of c-KIT were found in 14 sensitive tumors (P < 0.001), indicating the close association of c-KIT secondary mutations with imatinib-acquired resistance. In our study, 19 patients received sunitinib treatment after the failure of imatinib, and it seemed that the median progression-free survival (7 vs. 19 months, P = 0.244) in patients with secondary mutations (n = 13) was lower than that in patients without secondary mutations (n = 6). Secondary mutations of c-KIT were significantly associated with acquired resistance to imatinib in Chinese GIST patients, and whether secondary mutations of c-KIT could influence the efficacy of sunitinib needed to be further investigated.