Abstract Responding to a need to identify the mutational processes that are responsible for malignant transformation, highly accurate duplex sequencing (DS) was employed to monitor acquisition of context-dependent high-resolution mutational spectra (MS) during the process of hepatocarcinogenesis. Aflatoxin B1 (AFB1) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB1-DNA adducts in hepatocytes seeds a population of mutations, mainly G:C>T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Four-day old male mice were treated with AFB1 using a regimen that induced hepatocellular carcinoma within 72 weeks. The DS technology revealed a distinctive MS of AFB1 at 10 weeks after dosing, well before evidence of neoplasia. Importantly, 25% of all mutations were G>T in one trinucleotide context (5’-CGC-3’), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB1 exposure. At 72 weeks, livers were separated into tumor tissue and normal surrounding cellular fractions (primarily hepatocytes). The MS in normal cells surrounding the tumors revealed clear evidence of previous AFB1 exposure with predominantly G:C>T:A mutations and strong representation of the CGC>CTC hotspot. Analysis of tumor tissue revealed a more complex pattern than that observed in surrounding hepatocyte fraction; tumor MS were a composite of the 10-week spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. The 10-week AFB1-exposed mouse MS was then used as bait to computationally identify human HCC sequences that have similarity to it. This analysis revealed that the mouse spectrum has striking similarity to human HCCs from hepatitis B infected people who have the R249S mutation in P53. The data also provide insight into the molecular basis for the synergism between hepatitis B exposure and AFB1. The MS provided by DS technology provides a method to identify mutational processes in normal and tumor tissues enabling reconstruction of the molecular etiology of human hepatocarcinogenesis and other genetic diseases. Citation Format: Supawadee Chawanthayatham, Charles C. Valentine III, Bogdan I. Fedeles, Stephen L. Slocum, Gerald N. Wogan, Robert G. Croy, John M. Essigmann. Mutation spectra of aflatoxin B1 in vivo establish biomarkers of exposure for human hepatocellualr carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-229. doi:10.1158/1538-7445.AM2017-LB-229