The mechanism by which heterogeneous-sized circulating tumor cells (CTCs) in gastric cancer (GC) patients are resistant to the targeted therapy and/or chemotherapy remains unclear. This study investigated prognostic value and genomic variations of size-heterogenous CTCs, in an attempt to unravel the molecular mechanisms underlying the therapeutic resistance, which is relevant to poor prognosis in GC. Aneuploid CTCs, detected in 111 advanced GC patients, were categorized into small (≤white blood cell [WBC], 25.54%) and large (>WBC, 74.46%) cells. Pre-treatment patients possessing ≥3 baseline small CTCs with trisomy 8 (_SCTCs^tri) or ≥6 large multiploid CTCs (_LCTCs^multi) showed an inferior median progression-free survival. Moreover, the cut-off value of ≥6 _LCTCs^multi was also an effective prognosticator for poor median overall survival. Single cell-based DNA sequencing of 50 targeted CTCs indicated that _SCTCs^tri and _LCTCs^multi harbored distinct gene variations respectively. Mutations in the KRAS and Rap1 pathway were remarkably abundant in _SCTCs^tri, whereas several unique mutations in the MET/PI3K/AKT pathway and SMARCB1 gene were identified in _LCTCs^multi. Obtained results suggested that _SCTCs^tri and _LCTCs^multi exhibited different mechanisms to therapy resistance and correlated with patients’ poor outcome.