Dasatinib is a small molecule tyrosine kinase inhibitor with multiple targets including kit, PDGFR, and SRC. This prospective study evaluated the efficacy and safety of dasatinib as third-line treatment for gastrointestinal stromal tumors (GIST). The study enrolled adult patients (≥18 years of age) with histologically confirmed unresectable and/or metastatic GIST whose disease progressed despite imatinib and sunitinib therapy. Dasatinib (50 mg twice daily) was given orally for 1 week and escalated to 70 mg twice daily orally. The primary endpoint was to the 3-month progression-free survival (PFS) rate. Blood samples were acquired before dasatinib therapy for examination of gene mutations by next-generation sequencing (NGS). From May 2016 to June 2018, 58 patients from 9 Chinese medical centers were enrolled in this study. The 3-month PFS rate was 53.4% and the median overall survival (OS) was 14.0 months. Neither primary nor secondary gene mutations predicted the efficacy of dasatinib. Wild-type GIST patients had longer PFS (5.5 months). The most common adverse events were anemia, proteinuria, fatigue, neutropenia, and diarrhea. The concordance of KIT/PDGFRA mutation was 61.9% between tissue and peripheral blood samples and additional KIT mutations were detected in the peripheral blood samples in 28.6% of the patients. Some SNV and CNV such as ATRX, TP53, TEKT4, STK11, SDHC, and CDKN2C related to tumor signaling pathways were detected. Patients with TP53 mutations and SDHC and TMEM127 gene copy number loss had longer OS. Dasatinib has modest antitumor activity with tolerable toxicities in patients with metastatic GISTs who have failed imatinib and sunitinib therapy. © 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.