Cells within tumours have diverse genomes and epigenomes and interact differentially with their surrounding microenvironment generating intratumour heterogeneity, which has critical implications for treating cancer patients. Understanding the cellular and microenvironment composition and characteristics in individual tumours is critical to stratify the patient population that is likely to benefit from specific treatment regimens. Here, we will review the current understanding of intratumour heterogeneity at the genomic, epigenomic and microenvironmental levels. We will also discuss the clinical implications and the challenges posed by intratumour heterogeneity and evaluate noninvasive methods such as circulating biomarkers to characterize the cellular diversity of tumours. Comprehensive assessment of the molecular features of patients based on tumour specimen characterization (including intratumour spatial and temporal variations), ancillary noninvasive methods (such as circulating biomarkers and molecular imaging approaches) and the correct design of clinical trials are required to guide administration of targeted therapy and to control therapeutic resistance. Finding the means to accurately determine and effectively control tumour heterogeneity and translate these achievements into patient benefit are major goals in modern oncology.