Background: Fibroblast growth factor receptor (FGFR) isoform switching from FGFR2b to FGFR2c has been implicated in epithelial-to-mesenchymal transition and enhanced invasive potential in various malignancies and potentially be involved in resistance mechanisms to FGFR2b targeted therapy (e.g., bemarituzumab). This study sought to investigate the potential of respective FGFR2 isoform expression evaluation utilizing whole-transcriptome sequencing (WTS) and its correlation with therapeutic efficacy and clinical outcomes in patients with advanced G/GEJ cancer. Methods: Patients with advanced G/GEJ cancer who received systemic therapy at the National Cancer Center Hospital East between May 2021 and September 2023, and were enrolled in the immunological profiling study (UMIN000019129) and MONSTAR-SCREEN-2 (UMIN000043899) were included. FGFR2 protein and RNA expression were assessed via immunohistochemistry (IHC) and WTS with Caris MI Profile, respectively. FGFR2b and FGFR2c isoforms were distinguished by the detection of their respective splice junctions using WTS data. The FGFR2b/(2b+2c) ratio was stratified into FGFR2b-dominat and -containing subtypes using the median value. The log-rank test was used to discern differences in overall survival (OS). Results: Among the 129 patients, 21 (16.3%) exhibited FGFR2-positivity by IHC (staining intensity 2+ or 3+≥1% membranous staining of tumor cells), and a significant correlation was observed between FGFR2 IHC status and RNA expression levels (TPM: transcript per million) in WTS (FGFR2-positive, median TPM=11.1; FGFR2-negative, median TPM=4.7; P=0.002). The distribution of FGFR2b/(2b+2c) ratio (median, 0.89) was: 10.9% (0≤x<0.2), 5.4% (0.2≤x<0.4), 7.0% (0.4≤x<0.6), 12.4% (0.6≤x<0.8), 43.4% (0.8≤x<1.0), and 20.9% (x=1.0). Among 58 patients treated with 1^st line therapy, no significant difference in treatment response was observed between the FGFR2b-dominat (67.9%) and FGFR2c-containing subtypes (53.3%) (P=0.259). Overall, the FGFR2c-containing subtype was associated with significantly shorter OS compared to the FGFR2b-dominat subtype (median OS, 10.1 vs. 17.9 months; hazard ratio, 1.72; 95% confidence interval, 1.03-2.86; log-rank P=0.037). Conclusions: Our findings underscore the substantial concordance between IHC and WTS analysis of FGFR2, the feasibility of distinguishing FGFR2 isoforms using WTS, and the prognostic implications of FGFR2c isoform in patients with advanced G/GEJ cancer. Further analysis is warranted in patients receiving FGFR2b targeted treatment.