Background: Periampullary adenocarcinoma, including pancreatic cancer, is a heterogeneous group of tumors with dismal prognosis. The human epithelial growth factor receptor family consists of four closely related members EGFR (HER1) - HER4. HER-targeting drugs have a beneficial effect on survival in several types of cancer, but the benefits have been modest in pancreatic cancer and the prognostic value of HER-expression remains unclear. The aim of this study was therefore to examine the expression and prognostic value of EGFR, HER2 and HER3 in periampullary cancer, with particular reference to histological subtype. Methods: Immunohistochemical expression of EGFR, HER2 and HER3 was analyzed in tissue microarrays with 175 periampullary adenocarcinomas consecutively operated at the University hospitals of Lund and Malmö, Sweden, 2001 – 2011, with follow up data on RFS and OS for up to 5 years. Results: The expression of EGFR and HER2 was similar in pancreatobiliary (PB) and intestinal (I) type tumors but HER3 was more often highly expressed in I-type tumors (52% vs 21%, p < 0.001). In PB-type tumors there were no associations between expression of HERs and clinicopathological parameters or survival. In I-type tumors, high EGFR expression was associated with larger tumors and high expression of HER3 was inversely associated with growth in peripancreatic fat (p = 0.003) and T-stage (p = 0.006). HER2 expression was not associated with any clinicopathological parameter. In I-type tumors, high expression of EGFR was associated with shorter OS and RFS in univariable Cox regression analysis (HR = 2.74, CI 1.32-5.69 for OS and HR = 2.58, CI 1.23-5.38 for RFS) but not in multivariable analysis. HER2 expression was not prognostic. High HER3 expression was associated with a prolonged RFS in univariable (HR = 0.45, CI 0.21-0.95) but not in multivariable analysis. Conclusions: High EGFR expression is an unfavorable prognostic factor and high HER3 expression a favorable prognostic factor in I-type periampullary cancer. Expression of HERs in PB-type tumors had no evident prognostic value. The prognostic value of HERs in periampullary cancer thus differ by histological subtype.