Abstract Background Among women with an elevated risk of hereditary breast and ovarian cancer who previously tested negative for pathogenic mutations in BRCA1 and BRCA2, a subset remain at increased risk of having hereditary breast, ovarian or other cancers, and should be offered multi-gene panel testing. We tested three groups of women who were enrolled in the UCSF Cancer Genetics and Prevention Program: (i) 97 women with a personal history of bilateral breast cancer, (ii) 104 women with a personal history of breast cancer and a first-degree or second-degree relative with ovarian cancer, and (iii) 99 women with a personal history of ovarian, fallopian tube, or primary peritoneal cancer. All women previously tested negative for pathogenic BRCA1 and BRCA2 mutations by either limited or comprehensive testing. Methods We performed comprehensive next-generation sequencing using a panel of 19 genes developed by Color Genomics (a CLIA-certified laboratory) covering ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, NBN, PALB2, PMS2, PTEN, RAD51C, RAD51D, STK11, and TP53. Results Across the groups tested, 9% had pathogenic mutations in one or more of the genes analyzed (8% in genes other than BRCA1 and BRCA2). Among these women, Ashkenazi Jewish and Hispanic women had elevated mutation rates compared to those of other ethnicities. In addition, we identified two women with pathogenic mutations in two cancer susceptibility genes, which has significant implications for family testing. These results demonstrate the importance of genetic testing of genes other than BRCA1 and BRCA2. Conclusions Among women with an elevated risk of hereditary breast and ovarian cancer who have previously tested negative for BRCA1 and BRCA2 mutations, we propose that women with characteristics of any of the three groups above be considered for subsequent multi-gene panel testing. Additionally, ethnicity and the possibility of multiple mutations may be indications for additional testing in these women and in family members of carriers. Citation Format: Crawford BB, Adams SB, Sittler T, Van den Akker J, Chan SB, Leitner O, Ryan LN, Gil E, Van 't Veer LJ. Multi-gene panel testing for hereditary cancer predisposition in unsolved high risk breast and ovarian cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-08-02.