Despite the success of targeted therapies in the treatment of cancer, the development of resistance limits the ability to translate this method into a curative treatment. The mechanisms of resistance have traditionally been thought of as intrinsic (ie, present at baseline) or acquired (ie, developed after initial response). Recent evidence has challenged the notion of acquired resistance. Although cancers are traditionally thought to be clonal, there is now evidence of intra-tumour genetic heterogeneity in most cancers. The clinical pattern of acquired resistance in many circumstances represents outgrowth of resistant clones that might have originally been present in the primary cancer at low frequency but that have expanded under the selective pressure imposed by targeted therapies. Here, we describe the potential role of clonal heterogeneity in resistance to targeted therapy, discuss genetic instability as one of its causes, and detail approaches to tackle intra-tumour heterogeneity in the clinic.