Abstract Objectives Our study aimed to observe the distribution of putative stem cells in irreversible pulpitis and to investigate the expression of specific molecules. Subjects and Methods Extracted third molar teeth were collected and divided into two groups: the normal pulp group and inflamed pulp group. Real‐time PCR was applied to detect the expression of several embryonic and dentinogenic genes. The expression of mesenchymal cell markers (STRO‐1, CD90, and CD146) and stromal cell‐derived factor 1α (SDF‐1α)/CXC chemokine receptor 4 (CXCR4) proteins was examined by immunohistochemical analysis. Results The expression levels of most embryonic and dentinogenic genes were not statistically different between the two groups. Immunohistochemical analysis revealed that in inflamed pulp, cells with positive expression for STRO‐1, CD90, and CD146 mainly resided in two specific niches, both adjacent to inflammatory sites: one in the pulp core and another in the odontoblast layer. SDF‐1α‐ and CXCR4‐positive cells were significantly correlated with STRO‐1‐positive cells. Double immunofluorescence analysis indicated that STRO‐1‐positive cells overlapped with SDF‐1α‐ and CXCR4‐positive cells near the inflammatory site. Conclusions This study gave a direct observation of putative stem cells distributed in irreversible pulpitis and implied a role of SDF‐1α/CXCR4 signaling in stem cell‐based therapies for reparative dentinogenesis.