Pancreatic cancer is one of the most lethal malignancies, primarily due to late-stage diagnosis and limited therapeutic options. Cancer stem cells (CSCs) contribute to tumor heterogeneity, therapy resistance, and recurrence through activation of developmental pathways such as Hedgehog, Wnt, Notch, JAK-STAT, and Hippo. Identifying CSCs is therefore essential for understanding pancreatic ductal adenocarcinoma (PDAC) pathogenesis and advancing targeted therapies. This study compares the expression of key CSC-associated markers (CD44, CD117, OCT3/4, and c-Myc) in PDAC, fetal, and adult pancreas to elucidate CSC dynamics. Immunohistochemistry for CSC markers (CD44, CD117, OCT3/4, and c-Myc) was performed on PDAC tissues and control pancreatic samples (fetal pancreas 28–36 weeks and adult pancreas) to evaluate marker expression. Proliferative potential was assessed using CK7 and Ki-67 expression patterns. CD44 showed strong membranous and cytoplasmic expression in PDAC, moderate in fetal pancreas (epithelial/ductal regions), and minimal expression in adult tissue. CD117 was mainly restricted to stromal cells in PDAC, also present in fetal tissue but low in adults.c-Myc and Ki-67 were moderately expressed in PDAC, significantly higher than the control samples. CK7 demonstrated strong cytoplasmic staining in PDAC, moderate expression in adults, and weak expression in fetal samples. CD44 and c-Myc re-expression in PDAC supports their role as CSC-associated markers and potential drivers of tumor progression. CD117’s stromal localization suggests tumor–stroma interactions. These findings highlight developmental reactivation of CSC markers in PDAC, with implications for early detection and targeted therapy.