Direct sequencing can be used for characterizing mutagenicity in complex biological models, e.g., in various tissues of mammalian species. We employed whole-genome High-Fidelity Sequencing (HiFi Sequencing) for detecting mutations induced in male CD-1 mice by N-ethyl-N-nitrosourea (ENU). Mice were treated by gavage with a single 40 mg/kg dose of ENU at 12 weeks of age; negative control mice were untreated. Peripheral blood and solid tissues (liver, spleen, and kidney) were harvested 4 weeks post-exposure; the erythrocyte Pig-a assay was used to measure mutant frequencies in peripheral blood, while mutations were evaluated in the solid tissues by HiFi Sequencing. The frequency of Pig-a phenotypically mutant total red blood cells and reticulocytes in ENU-treated mice increased 7- and 30-fold, while the frequency of mutations in the genomic DNA of solid tissues increased up to 7-fold, with the greatest increase observed in the spleen and the smallest increase in the liver. The most common mutations detected by HiFi Sequencing in ENU-treated mice were T>A transitions and T>C transversions. The data suggest that HiFi Sequencing complements the Pig-a reporter gene mutation assay, detecting mutagenicity in tissues where performing the Pig-a assay is impossible and providing information on the spectra of mutations which potentially may be useful for characterizing the genotoxicity of novel compounds.