Background: DNA repair genomic alterations (GAs) have been identified in 12% of Intrahepatic cholangiocarcinoma (IHCCA), 26% of extrahepatic CCA (EHCCA) and 8% of gallbladder cancer (GBC) patients (pts) (Cancer 2016;122:3838–3847). Recently, the Cancer Genome Atlas (TCGA) has described more than 20 mutated DNA repair genes, many of which were not previously represented in prior reports. DNA repair GAs including MSI are associated with higher tumor mutational burden (TMB). Our study aim is to identify variations in the frequency of DNA repair GAs in IHCCA, EHCCA, and GBC. Methods: Hybrid capture-based next-generation sequencing of 422 fixed formalin paraffin embedded (FFPE) tissue blocks of BTC including; 270 IHCCA, 60 EHCCA, and 92 GBC was performed. We included 20 DNA repair genes and classified them as "direct" DNA repair genes (ATM, ATR, BRCA1, BRCA2, FANCA, FANCD2, MLH1, MSH2, MSH6, PALB2, POLD1, POLE, PRKDC, RAD50, SLX4) and "caretaker" genes that induce genomic instability (BAP1, CDK12, MLL3, TP53, BLM). We calculated TMB on 1.1 Mb of sequenced DNA in 205 tissue specimens [138 IHCCA, 23 EHCCA, and 44 GBC] and classified into three groups; high (TMB-H; ≥ 20 mut/Mb), intermediate (TMB-I; 6-19mut/Mb) and low (TMB-L; < 6mut/Mb). Results: A distinct pattern of DNA repair GAs in each tumor type was detected. (Table 1). DNA repair GAs were more commonly noted in both EHCCA and GB (63%) as compared with IHCAA (45.2%) (p= .002). Direct DNA repair GAs were highest within EHCCA (25%), while indirect DNA GAs were predominant in GBC (59.8%) (p= 0.04 and 0.001, respectively). The frequency of TMB-H and TMB-I differed significantly between BTC subtypes. Pts with EHCCA and GBC had significantly higher TMB-H and TMB-I versus IHCCA. Conclusions: The frequency of DNA repair GA’s is higher in EHCCA and GBC as compared with IHCCA. These results may have implications for clinical trials with DNA repair inhibitors and immune checkpoint blockers.