Purpose: In many children with cancer and characteristics suggestive of a genetic predisposition syndrome, the genetic cause is still unknown. We studied the yield of pathogenic mutations by applying whole-exome sequencing on a selected cohort of children with cancer. Experimental Design: To identify mutations in known and novel cancer-predisposing genes, we performed trio-based whole-exome sequencing on germline DNA of 40 selected children and their parents. These children were diagnosed with cancer and had at least one of the following features: ( 1 ) intellectual disability and/or congenital anomalies, ( 2 ) multiple malignancies, ( 3 ) family history of cancer, or ( 4 ) an adult type of cancer. We first analyzed the sequence data for germline mutations in 146 known cancer-predisposing genes. If no causative mutation was found, the analysis was extended to the whole exome. Results: Four patients carried causative mutations in a known cancer-predisposing gene: TP53 and DICER1 ( n = 3). In another 4 patients, exome sequencing revealed mutations causing syndromes that might have contributed to the malignancy ( EP300 -based Rubinstein–Taybi syndrome, ARID1A -based Coffin–Siris syndrome, ACTB -based Baraitser–Winter syndrome, and EZH2 -based Weaver syndrome). In addition, we identified two genes, KDM3B and TYK2 , which are possibly involved in genetic cancer predisposition. Conclusions: In our selected cohort of patients, pathogenic germline mutations causative or likely causative of the cancer phenotype were found in 8 patients, and two possible novel cancer-predisposing genes were identified. Therewith, our study shows the added value of sequencing beyond a cancer gene panel in selected patients, to recognize childhood cancer predisposition. Clin Cancer Res; 24(7); 1594–603. ©2018 AACR .