Objective The aim of this study was to investigate the association of TWIST1 and CD44 in gastric cancer (GC) with clinical parameters and their relation to prognosis, which may be beneficial for targeted therapeutic strategies in the future. Materials and methods The material of this work consisted of 40 primary GC specimens from patients who underwent radical gastrectomy. Patients who received neoadjuvant chemotherapy or chemoradiotherapy, those who presented with other cancers at the same time, or the patients with incomplete clinical data were excluded from the study. Hematoxylin and eosin-stained sections from all cases were re-evaluated and further stained immunohistochemically using antibodies against TWIST1 and CD44. Results TWIST1 and CD44-positive expressions were significantly increased in GC cases of diffuse type (P=0.019 and 0.002, respectively). Moreover, there was a statistically significant correlation between both markers and tumor grade, stage, and lymphovascular invasion (P=0.027 and 0.010, P=0.002 and 0.012, and P=0.001 and 0.005, respectively). A statistically significant correlation was found between TWIST1 and CD44 expressions in GCs (P=0.000). Conclusion The presence of TWIST1-positive carcinoma cells and CD44-positive cancer stem-like cells in GC tissue can be used as a diagnostic tool for GC and regarded as a marker of poor prognosis in patients with GC, which may provide potential targets for GC therapy.