We developed ‘3D Live-Seq’, a protocol that enables the reconstruction of evolving tumor cell karyotypes across multiple consecutive cell generations by integrating live-cell imaging data of tumor organoid outgrowth and whole-genome sequencing data of each imaged cell. Using patient-derived colorectal cancer organoids and freshly isolated tumor tissue, we demonstrate that chromosomal copy-number alterations of varying complexity are prevalent and can arise within a few cell generations.Here deposited are the discrete copy-number values per bin for each single cell or bulk sequenced sample.