CDK7 has emerged as a cancer target because of its pivotal roles in cell cycle progression and transcription. Several CDK7 inhibitors (CDK7i) are now in clinical evaluation. Identifying patients most likely to respond to treatment and early detection of tumour evolution towards resistance are necessary for optimal implementation of cancer therapies. Continuous culturing of prostate cancer cells with Samuraciclib, a non-covalent ATP-competitive CDK7i, led to outgrowth of resistant cells. These were characterised by the acquisition of a single base change in the CDK7 gene, Asp97 to Asn (D97N). Mutant cells were resistant to other non-covalent CDK7i but remained sensitive to covalent CDK7i. Cryo-EM structure and kinase ligand affinity determinations revealed reduced affinity of the CDK7-D97N mutant for non-covalent CDK7i. Remarkably, Asp97 is absolutely conserved in human CDKs, inferring its importance for the activities of all CDKs. Consistent with this, mutation of the homologous residue in CDK12 (D819N) or CDK4 (D99N) promoted resistance to drugs that inhibit these CDKs. Our findings reveal a general mechanism for acquired resistance with obvious implications for patients treated with CDK inhibitors.