Abstract: The visual signal processing in the retina requires the precise organization of diverse neuronal types working in concert. While single-cell omics studies have identified more than 120 different neuronal subtypes in the mouse retina1, little is known about their spatial organization. Here, we generated the first single-cell spatial atlas of the mouse retina using multiplexed error-robust fluorescence in situ hybridization (MERFISH). We profiled over 390,000 cells and identified all major cell types and nearly all subtypes through the integration with reference single-cell RNA sequencing (scRNA-seq) data. Our spatial atlas allowed simultaneous examination of nearly all cell subtypes in the retina, revealing 8 previously unknown displaced amacrine cell subtypes and establishing the first connection between the molecular classification of many cell subtypes and their spatial arrangement. Furthermore, we identified spatially dependent differential gene expression between subtypes, suggesting the possibility of functional tuning of neuronal types based on location. Data description: 1. VZA105a_integrated_368genes.h5ad This file contains the raw MERFISH count matrix for four samples with 368 gene features. The "sampleid" column represents the unique sample ID, while the "region" column corresponds to the tissue section ID. The "majorclass" and "subclass" columns indicate annotated retinal cell types. Finally, the "center_x" and "center_y" columns provide the coordinates of the cell centers. 2. VA45_integrated.h5ad This file contains the raw MERFISH count matrix for six samples with 500 gene features. The "sampleid" column represents the unique sample ID, while the "region" column corresponds to the tissue section ID. The "majorclass" and "subclass" columns indicate annotated retinal cell types. Finally, the "center_x" and "center_y" columns provide the coordinates of the cell centers. 3. merfish_impute.h5ad This file contains the imputed count matrix for ten samples. The "sampleid" column represents the unique sample ID, while the "region" column corresponds to the tissue section ID. The "majorclass" and "subclass" columns indicate annotated retinal cell types. Finally, the "center_x" and "center_y" columns provide the coordinates of the cell centers.