The chicken DT40 cell line is a widely used model system in the study of multiple cellular processes due to the efficiency of homologous gene targeting. The cell line was derived from a bursal lymphoma induced by avian leukosis virus infection. In this study we characterized the genome of the cell line using whole genome shotgun sequencing and single nucleotide polymorphism array hybridization. The results indicate that wild type DT40 has a relatively normal karyotype except for whole chromosome copy number gains, and no karyotype variability within stocks. In a comparison to two domestic chicken genomes and the Gallus gallus reference genome we found no unique mutational processes shaping the DT40 genome except for a mild increase in insertion and deletion events, particularly deletions at tandem repeats. We mapped coding sequence mutations that are unique to the DT40 genome; mutations inactivating the PIK3R1 and ATRX genes likely contributed to the oncogenic transformation. In addition to a known avian leukosis virus integration in the MYC gene we detected further integration sites that are likely to de-regulate gene expression. The new findings support the hypothesis that DT40 is a typical transformed cell line with a relatively intact genome, therefore it is well suited to the role of a model system for DNA repair and related processes. The sequence data generated by this study, including a searchable de novo genome assembly and annotated lists of mutated genes, will support future research using this cell line