Background: We report our large cohort of pediatric cancer patients undergoing prospective agnostic germline sequencing. Our dataset is a significant addition to the 1,573 children reported to date who have undergone agnostic germline sequencing in previous large sequencing studies, each with ascertainment bias. Methods: 676 patients with pediatric solid tumors underwent matched tumor-normal targeted DNA sequencing from July 2015 to February 2020. At least 76 genes associated with cancer predisposition were analyzed in the germline, and variants were classified per American College of Medical Genetics guidelines. Pathogenic and likely pathogenic (P/LP) variants were reported to patients/families, who were offered genetic counseling and cascade testing with screening recommendations and referral to a surveillance clinic as appropriate. Results: One or more P/LP variants were found in 17% (115/676) of individuals when including low, moderate and high penetrance mutations in recessive and dominant genes, or 12% (81/676) when including moderate and high penetrance mutations in dominant genes. P/LP variants were detected in 40% (21/53) of patients with retinoblastomas, 8% (13/161) with neuroblastomas/ganglioneuroblastomas, 13% (14/112) with brain/spinal tumors, 8% (20/245) with sarcomas, and 12% (13/105) with other solid tumors. The most frequent mutations were in RB1 (n = 28) and TP53 (n = 8) in patients with associated tumors. Of patients with moderate/high penetrance mutations, 30% (24/81) had unexpected tumor types, with potential therapeutic relevance in 58% (14/24) including BRCA1 n = 2, BRCA2 n = 3, RAD51D n = 1, ATM n = 1 MLH1 n = 1, MSH2 n = 1, MSH6 n = 1, PMS2 n = 3, and SUFU n = 1. Two patients received immunotherapy based on their germline finding. Conclusions: P/LP germline variants are frequently present in patients with pediatric cancer. We are contributing significantly to the cohort size of agnostic sequencing in pediatric cancers. Our experience is similar to other studies with a ~12% detection rate of moderate and high penetrance mutations. Moderate/high penetrance mutations were concordant with the patient’s cancer history in 70% of cases, higher than previously reported, likely due to an enrichment of retinoblastoma. While many mutations are identified in patients with associated tumor types, a large proportion of mutations are unexpected based on the patient’s history. Clinical actionability of these findings may include screening, risk reduction, family planning, and increasingly targeted therapies.