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Introduction Small bowel involvement in systemic sclerosis (SSc) often results in severe gastrointestinal (GI) complications, contributing to morbidity and mortality, with no treatments available. Biological processes underlying small bowel involvement are currently not understood but are crucial for developing targeted therapies. We aimed to characterize, for the first time, the molecular and cellular landscape of the duodenum across different disease stages.Material and Methods Gastroduodenal biopsies from ten SSc patients enrolled in the ReSScue clinical trial with moderate to severe lower GI symptoms (UCLA GIT score) were analyzed. Five patients had early disease (<3.5 years) and five late disease (>3.5 years). Formalin-fixed paraffin-embedded samples were processed using the Visium HD Spatial Gene Expression Reagent kit v1. Libraries were sequenced on the Illumina NovaSeq X and aligned with Space Ranger 3.1.3. Data analysis was performed using Seurat v5 and Banksy. Differentially expressed genes (DEG) between early and late SSc were identified with MAST (adjusted p < 0.05). Pathway overrepresentation analysis and gene set enrichment analysis were conducted using clusterProfiler with the Gene Ontology database. Module scores were generated for selected pathways.Results To assess all layers of the duodenum, we annotated six tissue domains: villi, crypts, lamina propria (LP), Brunner’s glands, muscularis mucosae and isolated lymphoid follicles. In early vs. late SSc, 471 DEGs were upregulated and 30 downregulated in villi (|log2 FC| > 0.4), 352 upregulated and 31 downregulated in crypts, and 61 upregulated and 2,355 downregulated in the LP (|log2 FC| > 0.2). Pathogen defense pathways were upregulated in all three domains in early SSc. T and B cell activation was elevated in villi and crypts of early patients, and in the LP during late disease. In contrast, extracellular matrix (ECM) organization was significantly upregulated in the LP of late SSc patients. Interestingly, we observed an upregulation of metabolic pathways across several tissue domains in late SSc. Cellular respiration was enriched in villi, lipid metabolism in crypts and LP, and antioxidant activity in the LP.Conclusions We show significant gene expression changes in different duodenal layers between early and late SSc, highlighting distinct biological processes at play during the disease course. While early disease is marked by epithelial immune activation and pathogen defense, late disease shows deeper immune infiltration, enhanced ECM remodeling, and metabolic reprogramming. These findings provide insight into the shifting biological processes in SSc progression of small bowel involvement and are crucial for developing targeted therapeutic strategies.