Secondary lymphoid organs (SLOs), including tonsils, lymph nodes, and Peyer’s Patches, exhibit complementary immune functions. However, little is known about the spatial organization of immune cells and extracellular matrix (ECM) in the SLOs. Traditional imaging is limited to a few markers, confining our understanding of the differences between the SLOs. Herein, Imaging mass cytometry (IMC) addressed this gap by simultaneously profiling a 25-plex proteins in SLO tissues at subcellular resolution. The antibody panel targeted immune, stromal, chemokine, epigenetic, and functional markers. For robust cell identification, a computational pipeline SpatialVizPheno was developed to spatially phenotype 999,970 cells using two approaches, including the manual gating and a semi-supervised gating, iterative clustering, and annotation. Lymph nodes exhibited the highest density of B cells while the intestinal tissues contained the highest proportion of regulatory and follicular helper T-cells. SpatialVizPheno identified the most prevalent interaction between follicular dendritic cells and stromal cells, plasma cells, and the stromal cells across the lymphoid tissues. Collagen-enriched regions were associated with the spatial orientation of B cell follicles in both tonsil and lymph node tissues, but not in intestinal lymphoid tissues. Such spatial differences of immunophenotypes and ECM in different SLO tissues can be used as a tool to quantify the relation of cellular organization and ultimate immune responses.