Abstract Polo-like kinase 1 (PLK1) is an essential cell-cycle regulator that is frequently overexpressed in various human cancers. To determine whether Plk1 overexpression drives tumorigenesis, we established transgenic mouse lines that ubiquitously express increased levels of Plk1. High Plk1 levels were a driving force for different types of spontaneous tumors. Increased Plk1 levels resulted in multiple defects in mitosis and cytokinesis, supernumerary centrosomes, and compromised cell-cycle checkpoints, allowing accumulation of chromosomal instability (CIN), which resulted in aneuploidy and tumor formation. Clinically, higher expression of PLK1 positively associated with an increase in genome-wide copy-number alterations in multiple human cancers. This study provides in vivo evidence that aberrant expression of PLK1 triggers CIN and tumorigenesis and highlights potential therapeutic opportunities for CIN-positive cancers. Significance: These findings establish roles for PLK1 as a potent proto-oncogene and a CIN gene and provide insights for the development of effective treatment regimens across PLK1-overexpressing and CIN-positive cancers.