Abstract Background Therapeutic options for neuroendocrine neoplasms (NEN) are limited, and only few NEN patients have been treated according to their individual genomic profile. We report the molecular and clinical characteristics of a large NEN cohort that was studied by prospective whole-genome (WGS), whole-exome (WES), and transcriptome sequencing (TS) within the NCT/DKTK MASTER precision oncology program. Methods Between 2013 and 2018, a total of 115 patients with advanced, heavily pretreated NEN were enrolled. Primary sites were gastrointestinal (n = 30), pancreatic/hepatic (n = 33), pulmonary (n = 20), genitourinary (n = 13), head/neck (n = 6), and other regions (n = 11). Grading according to the 2017 and 2019 WHO classification was neuroendocrine tumor (NET) G1, n = 12; NET G2, n = 31; NET G3, n = 12; neuroendocrine carcinoma, n = 36; mixed neuroendocrine-non-neuroendocrine neoplasm, n = 12; and other, n = 11. Results Of WES (n = 72) or WGS (n = 44) and TS (n = 92) were discussed in a multidisciplinary molecular tumor board. Recommendations were given in 103 cases (94%) for the following potential treatment alternatives: tyrosine or serine/threonine kinase inhibition (n = 67), PARP inhibition (n = 41), immunotherapy (n = 33), mTOR inhibition (n = 30), CDK4/6 inhibition (n = 19), MEK inhibition (n = 12), platinum-based chemotherapy (n = 7), BET inhibition (n = 7), anti-claudin18.2 (n = 5) or anti-DLL3 antibody treatment (n = 8). Somatic hypermutation (>10 mutations/megabase) was seen in 8.6% of patients. Treatment options could be implemented in 35 patients, of which 31 were evaluable for response: partial response, n = 10; mixed response, n = 1; stable disease, n = 11; progressive disease, n = 9; resulting in an overall response rate of 32% and a disease control rate of 71%. Conclusions Comprehensive genomic and transcriptomic characterization provides new insight into the molecular pathogenesis of NEN and creates therapeutic opportunities in a significant proportion of NEN patients who have exhausted standard treatment. Legal entity responsible for the study NCT Heidelberg & German Cancer Consortium. Funding German Cancer Research Center. Disclosure S. Kreutzfeldt: Research grant / Funding (institution), Travel / Accommodation / Expenses: PharmaMar; Honoraria (self): Novartis; Research grant / Funding (institution): Roche; Travel / Accommodation / Expenses: Pfizer. C.E. Heilig: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: PharmaMar; Honoraria (institution), Travel / Accommodation / Expenses: Lilly; Travel / Accommodation / Expenses: Novartis; Travel / Accommodation / Expenses: Celgene; Travel / Accommodation / Expenses: Teva; Travel / Accommodation / Expenses: BMS; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Pfizer. All other authors have declared no conflicts of interest.