Background Despite the efforts to describe the molecular landscape of esophageal adenocarcinoma (EAC) and its precursor lesion Barrett’s esophagus (BE), discrepant findings are reported. Here, we investigated the prevalence of selected genetic ( TP53 mutations and microsatellite instability (MSI) status) and epigenetic (DNA promoter hypermethylation of APC , CDKN2A , MGMT , TIMP3 and MLH1 ) modifications in a series of 19 non-dysplastic BE and 145 EAC samples. Additional biopsies from adjacent normal tissue were also evaluated. State-of-the-art methodologies and well-defined scoring criteria were applied in all molecular analyses. Results Overall, we confirmed frequent TP53 mutations among EAC (28%) in contrast to BE, which harbored no mutations. We demonstrated that MSI and MLH1 promoter hypermethylation are rare events, both in EAC and in BE. Our findings further support that APC , CDKN2A , MGMT and TIMP3 promoter hypermethylation is frequently seen in both lesions (21–89%), as well as in a subset of adjacent normal samples (up to 12%). Conclusions Our study further enlightens the molecular background of BE and EAC. To the best of our knowledge, this is one of the largest studies addressing a targeted analysis of genetic and epigenetic modifications simultaneously across a combined series of non-dysplastic BE and EAC samples. Supplementary Information The online version contains supplementary material available at 10.1186/s13148-022-01287-7.