Endometriosis is characterized by ectopic endometrial-like epithelium and stroma, of which molecular characteristics remain to be fully elucidated. We sequenced 107 ovarian endometriotic and 82 normal uterine endometrial epithelium samples isolated by laser microdissection. In both endometriotic and normal epithelium samples, numerous somatic mutations were identified within genes frequently mutated in endometriosis-associated ovarian cancers. KRAS is frequently mutated in endometriotic epithelium, with a higher mutant allele frequency (MAF) accompanied by arm-level allelic imbalances. Analyses of MAF, combined with multiregional sequencing, illuminated spatiotemporal evolution of the endometriosis and uterine endometrium genomes. We sequenced 109 single endometrial glands and found that each gland carried distinct cancer-associated mutations, demonstrating the heterogeneity of the genomic architecture of endometrial epithelium. Remarkable increases in MAF of mutations in cancer-associated genes in endometriotic epithelium suggest retrograde flow of endometrial cells already harboring cancer-associated mutations, with selective advantages at ectopic sites, leading to the development of endometriosis. Display omitted •Endometriosis and uterine endometrium exhibit cancer-associated somatic mutations•Clonal expansion of epithelial cells with cancer-associated mutations in endometriosis•Genomic architecture of epithelial cells in uterine endometrium is heterogeneous•Single endometrial glands carry distinct mutations in cancer-associated genes Suda et al. identify numerous cancer-associated mutations in epithelial cells from ovarian endometriosis and normal endometrium. They describe a heterogeneous and mosaic-like uterine endometrial epithelium, shaped by endometrial glands with distinct somatic mutations. They suggest clonal expansion of epithelial cells with cancer-associated mutations leads to the development of endometriosis.