This study enrolled 10 patients diagnosed with premalignant lesions and early-stage gastric cardia adenocarcinoma (GCA), confirmed through endoscopic examination. These patients were subjected to next-generation sequencing (NGS) using a customized 1123-gene panel to identify genetic alterations and signaling pathways. The results were compared to stage IIB to IV GCA samples from the cancer genome atlas (TCGA) and a cohort of Hong Kong patients. The study provides insights into the molecular drivers of GCA progression, with potential therapeutic implications. A total of 10 patients diagnosed with premalignant and early-stage GCA were subjected to NGS targeted 1123-panal testing. Genetic alterations characteristics and signaling pathways were defined and analyzed. These findings were compared with the mutation features of stage IIB to IV GCA samples from the TCGA and another GCA cohort of HongKong patients (HK cohort). Additionally, therapeutic implications were also evaluated. In premalignant lesions and early-stage GCA, driver genes, such as TP53, ARIDA and LRP1B were found to have high mutation rates and showed no significantly different in driver gene mutation and tumor mutational burden with stage IIB to IV GCA in both the HK and TCGA-GCA cohorts. However, EPHA2 showed a significantly higher mutation rate in premalignant and early-stage GCA compared to IIB to IV GCA. The majority of 10 cancer-related signaling pathways were found to be activated in premalignant and early-stage GCA. Furthermore, 80% patients had corresponding potential therapeutic inhibitors based on molecular mutation results in our cohort. Certain mutational characteristics involved in the occurrence and progression of GCA are already present in premalignant lesions and early-stage GCA, which can be assessed and prevented through early molecular testing. Additionally, EPHA2 mutations are more common in premalignant lesions and early-stage GCA, which provided potential biomarkers for the diagnosis and detection of premalignant lesions and early-stage GCA.