Esophageal adenocarcinoma is heterogeneous and studies have reviewed many important mutations that contribute to the pathogenesis of the cancer. These discoveries have helped paved the way into identifying new gene markers or gene targets to develop novel molecular directed therapy for better patient outcomes in esophageal adenocarcinoma. Despite the recent bloom in next-generation sequencing, Sanger sequencing still represents the gold standard method for the study of the driver genes in esophageal adenocarcinoma. This chapter focuses on the sequencing techniques in identification of single gene mutations.