: The effectiveness of neoadjuvant chemoradiotherapy (nCRT) is variable in locally advanced rectal cancer (LARC) patients, the ypT3 stage having a minimal or moderate response. The aim of our study was the evaluation of the association between CD133 (Prominin1) and CD166 (ALCAM) expression, survival parameters, and clinicopathological characteristics of a subgroup of LARC patients who achieved ypT3, showing post-nCRT and TME tumor fragmentation response and the assessment of these CSCs biomarkers value as indicators of the nCRT tumor response. : Our study group comprised 60 LARC patients who achieved ypT3 status and exhibited a tumor fragmentation pattern following nCRT. Clinicopathological parameter and survival evaluations, along with CD133 and CD166 immunohistochemistry and scoring, were performed and the associations between different parameters were tested. : High CD133 expression was significantly associated with ypN category ( = 0.018), lymphovascular invasion (LVI) ( = 0.009), perineural invasion (PnI) ( = 0.006), and tumor grading ( = 0.047), while high CD166 expression was significantly associated with LVI ( = 0.020) and PnI ( = 0.028). Tumors with high CD133 and CD166 expressions were associated with decreased overall survival (OS) ( = 0.004 and = 0.006). Cox regression analysis identified high CD133 and CD166 expression as independent factors associated with reduced survival (HR = 3.237, = 0.014 and HR = 2.866, = 0.020). : Our results support the hypothesis that CD133 and CD166 are putative CSC biomarkers associated with aggressive behavior and a poor prognosis in LARC, offering opportunities for personalized targeted therapies.