Background Periampullary carcinoma is a heterogeneous group of malignancies, and despite advances in treatment, its mortality rate remains high. A better understanding of the disease and factors influencing its course and potential therapeutic targets is imperative for improving its overall outcome. Through comprehensive cytogenetic analysis, it has been established that the development of periampullary carcinogenesis involves specific chromosomal aberrations, dysregulation of oncogenes, and suppression of genes in a multistep progressive manner. Our study aimed to evaluate the expression of human epidermal growth factor (HER2Neu) in periampullary cancers using immunohistochemistry and fluorescent in situ hybridization. Material and methods This was a retrospective study in which all consecutive cases of periampullary carcinoma diagnosed over a period of three years were evaluated. HER2neu expression was analyzed using immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH). Histopathological evaluation was performed according to the College of American Pathologists (CAP) protocol. Results Twenty patients were diagnosed during the study period. On histomorphologic analysis, most cases (n=17) were diagnosed as well-differentiated adenocarcinomas, the most common subsite being the ampulla of Vater and pathological staging as pT2N0Mx. On IHC, no overexpression of HER2Neu was reported in any case, but FISH analysis revealed one point of amplification with HER/centromere enumerator probe (CEP) ratio>2. Conclusion HER2Neu evaluation in periampullary carcinoma has limited value; thus, it could have a restricted therapeutic role.