Abstract Chromosomal rearrangements and copy number aberrations are a major mechanism for driver gene alterations in breast cancer. However, the origins of chromosomal instability during breast tumorigenesis are poorly understood. We have recently shown that oncogene expression in normal mammary epithelial cells induces replication stress and a DNA damage response (DDR). The double-strand break sensor Mre11 is required for the oncogene-induced DDR, and suppression of Mre11 is sufficient to promote the development of Her2/Neu-driven breast cancer in a transgenic mouse model. Here we have applied single-cell genomics to primary mammary epithelial cells from this mouse model and demonstrate a profound effect of oncogene expression on chromosomal instability in preneoplastic cells, which is further augmented in Mre11-mutant cells. Her2/Neu-induced chromosomal breakpoints are highly enriched in the vicinity of large genes, suggesting a possible role for gene transcription and/or R-loops in the origin of oncogene-induced genomic fragility. Mre11 hypomorphism does not affect the number of chromosomal breakpoints but alters the genomic aberration signature to one that is over-represented by larger-size deletions. These findings indicate that chromosomal instability is an early event during Her2/Neu-initiated breast neoplasia. Furthermore, DDR perturbation—in the form of Mre11 hypomorphism—is not required for oncogene-induced genomic instability but rather alters the pattern of observed copy number aberrations and promotes the proliferative expansion of genomically unstable clones. Thus, single-cell genomics applied to murine models of breast preneoplasia reveals insights into the origins of chromosomal instability in breast cancer that may inform novel strategies to prognosticate or prevent the progression of high-risk premalignant breast lesions. Citation Format: Katerina Fagan-Solis, Dennis A. Simpson, Luciano Martelotto, Jorge S. Reis-Filho, John H. Petrini, Gaorav P. Gupta. Early onset of chromosomal instability in breast preneoplasia detected by single-cell genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3376.