Due to its larger surface area and ease of preparation, pig esophageal mucosa has emerged as a practical and viable alternative to buccal mucosa for studying permeability characteristics in vitro. However, the permeation results in biological membranes have shown high variability due to inter-specimen and intra-specimen variations. These biological variables can significantly influence the drug permeation across porcine esophageal mucosa. Therefore, this study aimed to investigate the viability associated with drug permeation by examining four to five porcine specimens (inter-specimen variations) and different regions of the esophageal mucosa within the same pig (intra-specimen variation). The analysis revealed that inter-specimen variation was more pronounced in the lipophilic model diffusant hydrocortisone, compared to the hydrophilic diffusant caffeine. Both model diffusants exhibited a linear increase in permeability from the upper esophageal sphincter (UES) to the lower esophageal sphincter (LES). Notably, the permeability of LES was 1.7-fold greater for caffeine and 2.5-fold greater for hydrocortisone compared to UES. The histological examination revealed that the epithelial thickness of UES was greater than the LES. Additionally, the lag time of both model diffusants decreased linearly from UES to LES. Based on these results, it could be concluded that intra-specimen variation is highly pronounced for hydrocortisone than caffeine. These findings contribute to the advancement of research in drug permeation studies, emphasizing the importance of carefully considering both inter-specimen variation and intra-specimen variation when utilizing porcine esophageal mucosa as an ex vivo model to human buccal mucosa.