Aims: Knowledge regarding the genetic features of ampullary carcinoma (AC) in European patients is limited. The utility of tumour markers for the establishment of a malignant diagnosis in biopsies from the ampullary region has not been fully elucidated. We aimed to describe the clinical, pathological, immunohistochemical (IHC) and genetic features of a Danish series of surgically resected ACs. Methods: Surgically resected ACs (n=59) were examined regarding (1) clinicopathological features, (2) histological subtypes, (3) expression of IMP3, maspin, MUC5AC and S100P and (4) next-generation sequencing using a hybrid capture-based platform (Illumina HiSeq2500), including 315 cancer-related genes plus introns from 28 genes often rearranged or altered in cancer. Tumour mutational burden (TMB) and microsatellite instability (MSI) were also evaluated. Results: Pancreatobiliary adenocarcinomas (PB-AC), intestinal adenocarcinomas (INT-AC), other ampullary tumours and mixed adenocarcinomas represented 45.8%, 23.7%, 16.9% and 13.6%. The proportion of IHC-positive ACs (score ≥2) was: Maspin (94.9%), IMP3 (67.8%), S100P (39.0%) and MUC5AC (18.6%). Most frequently altered genes were TP53 (59.3%), KRAS (40.7%), APC (27.8%), SMAD4 (20.4%), CDKN2A (16.7%) and ARID2/PIK3CA (each 11.1%). MUC5AC and S100P were frequently expressed in PB-AC, APC alterations frequent in INT-AC, SOX9 alterations were exclusive in INT-AC and MDM2 and FRS2 alterations in PB-AC. Four of 49 ACs (8.2%) were TMB-high/MSI-high and showed loss of MLH1 and PMS2. Conclusions: PB-AC was the most frequent histological subtype of AC. Maspin and IMP3 were the IHC tumour markers with the highest sensitivity. Adenocarcinoma subtypes differed regarding several genetic alterations, whose predictive value remains to be evaluated.