Abstract: Tumor heterogeneity is a common and important feature in the process of tumor evolution, supporting both tumor formation and further evolution. Although advances have been made in understanding the internal laws and formation mechanisms of tumor heterogeneity, tumor heterogeneity remains a major challenge to achieving an accurate clinical diagnosis, treatment plan, and overcoming drug resistance. With the development and continuous improvement of high-throughput sequencing and single-cell sequencing technology, the spatiotemporal variation and molecular mechanisms of tumor heterogeneity have been increasingly elucidated. Here, we systematically review the latest research progress in the complexity and formation mechanism of tumor heterogeneity, the effect that tumor heterogeneity has on tumor evolution, and its challenges in relation to clinical diagnosis and treatment.