Recent emergence of low-cost, high-throughput single-cell RNA-Sequencing technology (scRNA-Seq) has enabled systematic identification of new cell types. Molecular definition of cell types provides a way to classify cells and survey cellular heterogeneity. Establishing the spatial organization of cell types identified by scRNA-Seq analysis is essential and requires mapping of these cell types in three-dimensional (3D) tissue volumes. Here, we developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression and molecular cell types in thick tissue. We also developed a turnkey, open-source computational pipeline that allows for rapid and automated data processing, facilitating adoption of high-plex FISH in thick tissue as a routine laboratory method.