Background: Cancers of the pancreas and biliary tract remain one of the unfavorable malignant tumors with few driver genomic alterations. Tumor-agnostic approaches are promising for cancers with poor prognosis, with some potentially actionable alterations, such as BRCA1/2 mutations, ERBB2 amplification, MSI-High, or tumor mutational burden (TMB)-High. However, co-existing alterations, clinical significance of other genomic alterations, or frequency of alterations by clinical and genomic background are unclear. Here we investigated the genomic profile in a large cohort of advanced pancreatobiliary cancers to help refine and discover new targets for improved cancer therapies. Methods: Comprehensive genomic profiling was performed at Foundation Medicine, on patients with RWD tested during the course of routine clinical care. Hybrid capture was carried out on up to 395 cancer-related genes and select introns from up to 31 genes frequently rearranged in cancer. 16,913 pancreatic cancer (PC) patients and 3,031 biliary tract cancer (BTC) patients were available for analyses and were stratified by age (≥40/ < 40), MSI status, TMB status (High ≥10/Low < 10 Muts/Mb), and select gene alterations. Using a chi-square test with Yate’s correction, frequencies of genetic alterations were analyzed according to clinical or genomic background. Results:KRAS (84.8%), TP53 (73.3%), CDKN2A (51.2%), CDKN2B (26.5%), and SMAD4 (23.2%) were frequently altered in PC patients, versus TP53 (60.6%), CDKN2A (33.5%), KRAS (27.1%), CDKN2B (20.6%) and SMAD4 (16.9%) in BTC patients. The frequency of MSI-High and TMB-High in BTC was 1.2% and 5.7%, respectively, while these were lower in PC (0.48% and 2.1%, respectively). In PC patients, the KRAS alteration rate was significantly lower in both MSI-High (57.3%, P< 0.001) and TMB-High populations (51.3%, P< 0.001). In BTC patients, the rate of ERBB2 amplification was 6.4% in TMB-High and 8.6% in TMB-Low population. Interestingly, CDK12 rearrangement was observed in BTC patients with ERBB2 amplified tumors but not in those without ERBB2 amplified tumors. In patients of pediatric/adolescents and young adults ( < 40 years old), the mutation rate of KRAS/TP53/CDKN2A/SMAD4 was lower, and FGFR2 rearrangement (4%) was observed in PC patients; GATA6 amplification (11.1%) and rearrangement of BRAF (2.8%), FGFR2 (5.6%) were observed in BTC patients. Conclusions: A large real-world dataset showed differences in genomic landscape according to clinical or genomic background, and some potential targets for the development of novel drugs in advanced pancreatobiliary cancers. These findings may lead to the improvement of cancer therapies in PC and BTC patients with poor prognosis.