Barrett’s esophagus (BE) is considered a precancerous condition increasing the risk of esophageal adenocarcinoma (EAC) development. This study aimed to find biomarkers with the potential for differential diagnostics of BE and EAC. This pilot study comprised 24 endoscopically examined subjects, namely 12 patients with BE and 12 with EAC. Paired esophageal tissue samples (with the main pathology and adjacent tissue, paraffin-embedded) were histopathologically examined and the presence of CDX2, a diagnostic biomarker for BE//EAC, was immunohistochemically determined using a specific antibody. RNA was isolated from the paired fresh-frozen esophageal tissues, RNAseq library was prepared, and a single-read RNAseq (1x75) was conducted using an Illumina NovaSeq 6000 System. After rRNA removal and mapping to human reference, we obtained 2x 27.5-184 mil. reads per sample. Compared to EAC tissues, we observed a downregulation of the hallmark pathway for angiogenesis and an upregulated hallmark pathway for bile acid metabolism in BE (p<0.01). CDX2 protein and CDX2 gene were highly expressed in tissues with the main pathology in comparison to the adjacent tissue from both BE and EAC patients (p<0.001). On the other hand, the expression of MUC2 (mucin 2) as well as ACER2 (alkaline ceramidase 2) were upregulated in the BE tissue, and among others, TFPI2 (tissue factor pathway inhibitor 2) was downregulated in BE vs EAC tissues (p<0.001). In line with the literature, we confirmed that MUC2 is expressed in BE but not in EAC tissues. It has, therefore, the potential to serve as a biomarker of both differential diagnosis and/or prognosis.