Simple Summary: The molecular heterogeneity of esophageal adenocarcinoma (EAC), a severe malignancy with increasing incidence and low survival rates, misperceives the underlying biology of tumor onset and development. However, advances in high-throughput next-generation sequencing (NGS) technologies have highlighted the potential role of somatic DNA sequence markers for new diagnostic techniques or constitute novel therapeutic targets. Thus, in order to identify a molecular and prognostic signature in EAC patients, we decided to integrate the sequencing of specimens from naïve patients (not treated with chemo-radiotherapy) with histological classification, with the aim of identification of potential biomarkers, and patient stratification. Combining different approaches paves the way for early identification and the selection of better therapy. Esophageal adenocarcinoma (EAC) is a severe malignancy with increasing incidence, poorly understood pathogenesis, and low survival rates. We sequenced 164 EAC samples of naïve patients (without chemo-radiotherapy) with high coverage using next-generation sequencing technologies. A total of 337 variants were identified across the whole cohort, with TP53 as the most frequently altered gene (67.27%). Missense mutations in TP53 correlated with worse cancer-specific survival (log-rank p = 0.001). In seven cases, we found disruptive mutations in HNF1alpha associated with other gene alterations. Moreover, we detected gene fusions through massive parallel sequencing of RNA, indicating that it is not a rare event in EAC. In conclusion, we report that a specific type of TP53 mutation (missense changes) negatively affected cancer-specific survival in EAC. HNF1alpha was identified as a new EAC-mutated gene. [ABSTRACT FROM AUTHOR]