Basement membranes (BMs) are complex macromolecular networks underlying all continuous layers of cells. Essential components include collagen IV and laminins, which are affected by human genetic variants leading to a range of debilitating conditions including kidney, muscle, and cerebrovascular phenotypes. We investigated the dynamics of BM assembly in human pluripotent stem cell-derived kidney organoids. We resolved their global BM composition and discovered a conserved temporal sequence in BM assembly that paralleled mammalian fetal kidneys. We identified the emergence of key BM isoforms, which were altered by a pathogenic variant in COL4A5. Integrating organoid, fetal and adult kidney proteomes we found dynamic regulation of BM composition through development to adulthood, and with single-cell transcriptomic analysis we mapped the cellular origins of BM components. Overall, we define the complex and dynamic nature of kidney BM assembly and provide a platform for understanding its wider relevance in human development and disease.