BACKGROUND Spatial transcriptomics has greatly contributed to our understanding of spatial and intra-sample heterogeneity, which could be crucial for deciphering the molecular basis of human diseases. Intra-tumor heterogeneity, for example, may be associated with cancer treatment responses. However, the lack of computational tools for exploiting cross-regional information and the limited spatial resolution of current technologies present major obstacles to elucidating tissue heterogeneity. RESULTS To address these challenges, we introduce RegionalST, an efficient computational method that enables users to quantify cell type mixture and interactions, identify sub-regions of interest, and perform cross-region cell type-specific differential analysis for the first time. Our simulations and real data applications demonstrate that RegionalST is an efficient tool for visualizing and analyzing diverse spatial transcriptomics data, thereby enabling accurate and flexible exploration of tissue heterogeneity. Overall, RegionalST provides a one-stop destination for researchers seeking to delve deeper into the intricacies of spatial transcriptomics data. BACKGROUND The implementation of our method is available as an open-source R/Bioconductor package with a user-friendly manual available at https://bioconductor.org/packages/release/bioc/html/RegionalST.html. BACKGROUND Supplementary materials are available at Bioinformatics online.