Background & Aims We investigated the prevalence of germline mutations in APC , ATM , BRCA1 , BRCA2 , CDKN2A , MLH1 , MSH2 , MSH6 , PALB2 , PMS2 , PRSS1 , STK11 , and TP53 in patients with pancreatic cancer. Methods The Ontario Pancreas Cancer Study enrolls consenting participants with pancreatic cancer from a province-wide electronic pathology database; 708 probands were enrolled from April 2003 through August 2012. To improve the precision of BRCA2 prevalence estimates, 290 probands were selected from 3 strata, based on family history of breast and/or ovarian cancer, pancreatic cancer, or neither. Germline DNA was analyzed by next-generation sequencing using a custom multiple-gene panel. Mutation prevalence estimates were calculated from the sample for the entire cohort. Results Eleven pathogenic mutations were identified: 3 in ATM , 1 in BRCA1 , 2 in BRCA2 , 1 in MLH1 , 2 in MSH2 , 1 in MSH6 , and 1 in TP53 . The prevalence of mutations in all 13 genes was 3.8% (95% confidence interval, 2.1%−5.6%). Carrier status was associated significantly with breast cancer in the proband or first-degree relative ( P < .01), and with colorectal cancer in the proband or first-degree relative ( P < .01), but not family history of pancreatic cancer, age at diagnosis, or stage at diagnosis. Of patients with a personal or family history of breast and colorectal cancer, 10.7% (95% confidence interval, 4.4%−17.0%) and 11.1% (95% confidence interval, 3.0%−19.1%) carried pathogenic mutations, respectively. Conclusions A small but clinically important proportion of pancreatic cancer is associated with mutations in known predisposition genes. The heterogeneity of mutations identified in this study shows the value of using a multiple-gene panel in pancreatic cancer.