Mutations of mtDNA are an important cause of human disease, but few animal models exist. Because mammalian mitochondria cannot be transfected, the development of mice with pathogenic mtDNA mutations has been challenging, and the main strategy has therefore been to introduce mutations found in cell lines into mouse embryos. Here, we describe a phenotype-driven strategy that is based on detecting clonal expansion of pathogenic mtDNA mutations in colonic crypts of founder mice derived from heterozygous mtDNA mutator mice. As proof of concept, we report the generation of a mouse line transmitting a heteroplasmic pathogenic mutation in the alanine tRNA gene of mtDNA displaying typical characteristics of classic mitochondrial disease. In summary, we describe a straightforward and technically simple strategy based on mouse breeding and histology to generate animal models of mtDNA-mutation disease, which will be of great importance for studies of disease pathophysiology and preclinical treatment trials. Display omitted •We present a method to isolate and identify pathogenic mtDNA mutations in mice•We describe a mouse with a pathogenic mutation in the mitochondrial tRNAALA gene•The mice display disrupted mitochondrial translation as a result of the mutation•The mice display molecular and histochemical symptoms of human mitochondrial disease Kauppila et al. describe a phenotype-based screen in live mice to generate mouse models with pathogenic mtDNA mutations. As proof of concept, they present a mouse with a mutation in the mitochondrial tRNAALA gene that displays molecular and histochemical symptoms of human mitochondrial disease.