Abstract Chemotherapies are often given without precision biomarkers, exposing patients to toxic side effects without guaranteed benefit. Here we present chromosomal instability signature biomarkers that identify resistance to platinum-, taxane- and anthracycline-based treatments using a single genomic test. In retrospectively emulated randomized-control biomarker clinical trials using real-world cohorts (n = 840), predicted resistant patients had elevated treatment failure risk for taxane (hazard ratio (HR) of 7.44) and anthracycline (HR of 1.88) in ovarian, taxane (HR of 3.98) and anthracycline (HR of 3.69) in metastatic breast and taxane (HR of 5.46) in metastatic prostate. Nonrandomized emulations showed predictive capacity for platinum resistance in ovarian (HR of 1.46) and anthracycline in sarcoma (HR of 3.59). We demonstrate feasibility using whole-genome sequencing, capture-panel sequencing and cell-free DNA. Our findings highlight the clinical value of chromosomal instability signatures in predicting resistance to chemotherapies across multiple cancer types, with the potential to transform the one-size-fits-all chemotherapy approach into precise, tailored treatment.