Gastric cancer (GC) is one of the most common malignancies worldwide. Recently, cancer stem cells (CSCs) in tumors were found to possess the ability to sustain tumor self-renewal, initiate tumor progression, and possibly also contribute to cancer metastasis. We immunohistochemically examined expression and distribution of representative CSC markers ALDH1, CD44, and CD133 in primary tumors and lymph node metastasis of GC. Among 190 GC primary tumors, 104 (55%) were positive for ALDH1, 117 (62%) were positive for CD44, and 18 (9%) were positive for CD133. Expression of these three CSC markers was significantly associated with advanced clinicopathologic factors. Patients with CD44- and CD133-positive GC had a poorer survival rate than patients with CD44- and CD133-negative GC (CD44: P < 0.001, CD133: P= 0.006). Univariate and multivariate Cox proportional hazards analysis revealed tumor node metastasis stage, CD44 expression, and CD133 expression to be independent predictors of survival in patients with GC. Comparison of CSC markers in primary and metastatic sites showed ALDH1 positivity to be significantly higher in diffuse-type lymph node metastasis than in the primary tumor (P < 0.001). These results indicate that these CSC markers are important in tumor invasion and metastasis and may be good markers indicating long-term survival in patients with GC.