Genetic mosaicism is the result of the accumulation of somatic mutations in the human genome starting from the first postzygotic cell generation and continuing throughout the whole life of an individual. The rapid development of next-generation and single-cell sequencing technologies is now allowing the study of genetic mosaicism in normal tissues, revealing unprecedented insights into their clonal architecture and physiology. The somatic variant repertoire of an adult human neuron is the result of somatic mutations that accumulate in the brain by different mechanisms and at different rates during development and ageing. Non-pathogenic developmental mutations function as natural barcodes that once identified in deep bulk or single-cell sequencing can be used to retrospectively reconstruct human lineages. This approach has revealed novel insights into the clonal structure of the human brain, which is a mosaic of clones traceable to the early embryo that contribute differentially to the brain and distinct areas of the cortex. Some of the mutations happening during development, however, have a pathogenic effect and can contribute to some epileptic malformations of cortical development and autism spectrum disorder. In this Review, we discuss recent findings in the context of genetic mosaicism and their implications for brain development and disease.