Abstract Background Pediatric cancers are the leading cause of death by disease in children despite improved survival rates overall. The contribution of germline genetic susceptibility to pediatric cancer survivors has not been extensively characterized. We assessed the frequency of pathogenic/likely pathogenic (P/LP) variants in 5,451 long-term pediatric cancer survivors from the Childhood Cancer Survivor Study. Methods Exome sequencing was conducted on germline DNA from 5,451 pediatric cancer survivors (cases that survived ≥5 years from diagnosis; n = 5,105 European) and 597 European cancer-free adults (controls). Analyses focused on comparing the frequency of rare P/LP variants in 237 cancer-susceptibility genes, and a subset of 60 autosomal dominant high-to-moderate penetrance genes, for both case-case and case-control comparisons. Results 4.1% of European cases harbored a P/LP variant in high-to-moderate penetrance autosomal dominant genes, compared to 1.3% in controls (two-sided p = 3 x 10-4). The highest frequency of P/LP variants were in genes typically associated with adult-onset rather than pediatric cancers, including BRCA1/2, FH, PALB2, PMS2 and CDKN2A. A statistically significant excess of P/LP variants, after correction for multiple tests, were detected in patients with central nervous system cancers (NF1, SUFU, TSC1, PTCH2), Wilms tumor (WT1, REST), non-Hodgkin lymphoma (PMS2), and soft tissue sarcomas (SDHB, DICER1, TP53, ERCC4, FGFR3), compared to other pediatric cancers. Conclusion In long-term pediatric cancer survivors, we identified P/LP variants in cancer-susceptibility genes not previously associated with pediatric cancer as well as confirmed known associations. Further characterization of variants in these genes in pediatric cancer will be important to provide optimal genetic counseling for patients and their families.