The spatial organization of cells within tissues is tightly linked to their biological function. Yet, methods to probe the entire transcriptome of multiple native tissue microenvironments at single cell resolution are lacking. Here, we introduce fragment-sequencing, a method that enables the transcriptomic characterization of single cells within spatially distinct tissue niches. Fragment-sequencing of the mouse metastatic liver revealed previously uncharacterized zonated genes and ligand-receptor interactions enriched in the different hepatic microenvironments and the metastatic niche. Overall design: Method development of fragment-sequencing (based on single cell RNA sequencing and cell hashing) and its application to metastatic mouse liver (9 replicates), untreated mouse liver (1 replicate), untreated mouse spleen (2 replicates), Crohn?s disease biopsies (2 replicates) and a mix of human and mouse colorectal cancer organoids (1 replicate). To compare Visium of metastatic mouse livers (2 replicates) and conventional scRNAseq of metastatic mouse livers (2 replicates) were performed. Please note that the records have been updated with two additional samples on Feb 6th, 2023. Human samples were uploaded August 7th, 2023 and raw data were not provided due to patient privacy concerns.