Tissue architecture is a product of a multilayered molecular landscape, where even subtle disruptions in the spatial context can initiate or reflect disease processes. Recent advances in high-throughput spatial omics technologies have enabled the investigation of this complexity in stunning detail, providing groundbreaking insights into how spatial molecular organization shapes health and disease. Spatial analysis empowers the discovery of developmental and disease-associated molecular signatures, cell states and multicellular niches, as well as the evaluation of disease heterogeneity within and across organs. This Review examines spatially resolved pathological molecular alterations in a wide range of disease processes, such as developmental disorders, tumorigenesis, fibrosis and injury responses, neurodegeneration, infection and inflammation, through the lens of these universal biological frameworks. We discuss challenges, opportunities and promising examples in advancing these technologies to clinical applications, including the increasing importance of artificial intelligence. Finally, we explore possible avenues for a more comprehensive, multidimensional assessment of tissues.