BACKGROUND: This study aimed to perform a comprehensive gene expression analysis in patients with early-stage gastric cancer (EGC) to identify gene expression profiles specific to gastric cancer (GC) lesions. METHODS: Biopsy specimens were collected from one EGC lesion and three background mucosal areas of patients scheduled for endoscopic submucosal dissection (ESD). Lesion-specific gene profiles in these four biopsies were analyzed using DNA microarrays. Patients with concurrent EGCs at the time of an ESD or a history of GC were classified into the multiple GC group ( RESULTS: After excluding patients with heterogeneous factors, 55 patients were analyzed. Twenty-one differential genes exhibiting distinct mean expression profiles stratified by background gastric mucosa were extracted between the single and multiple GC groups. A scoring system constructed using these genes to calculate the weighted expression values for each patient, with an optimal cutoff value of -2.574, yielded a sensitivity and specificity of 85.7%. CONCLUSIONS: This study identified the different gene expression profiles between synchronous and metachronous multiple GCs and single GCs in patients with EGC. The developed scoring system has potential to distinguish between single and multiple GCs.