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Analyzing multi-sample spatial transcriptomics data requires accounting for biological variation. We present multi-sample non-negative spatial factorization (mNSF), an alignment-free framework extending single-sample spatial factorization to multi-sample datasets. mNSF incorporates sample-specific spatial correlation modeling and extracts low-dimensional data representations. Through simulations and real data analysis, we demonstrate mNSF’s efficacy in identifying true factors, shared anatomical regions, and region-specific biological functions. mNSF’s performance is comparable to alignment-based methods when alignment is feasible, while enabling analysis in scenarios where spatial alignment is unfeasible. mNSF shows promise as a robust method for analyzing spatially resolved transcriptomics data across multiple samples.