Cancer stem cells (CSCs) are proposed to be involved in colorectal cancer (CRC) initiation, growth, and metastasis. The aim of our pilot study was to assess possible correlations between the clinicopathological characteristics of CRC patients and CSCs gene expression patterns, in order to provide insight into new methods for patient stratification and targeted therapeutic strategies. Our study involved 60 CRC patients, and the following three specific CSC genes were targeted: PROM1/CD133, ALCAM/CD166 and HCAM /CD44. Data are presented as relative mRNA expression of target genes to GAPDH. The expression of total CD133 and CD166 was assessed in paired samples of CRC tumors and adjacent tissue, while CD44 was assessed in similar samples. The qRT-PCR analysis detected all three targeted genes to different extents, in both normal and tumor tissue. In nine cases (15.69%), total CD133 had a higher expression in tumor tissue, whilst in 28 cases (47.06%) the expression was higher in non-malignant peritumor tissue. The total CD166 expression was increased in tumor tissue compared with paired non-invaded peritumor samples in eight cases (13.73%), whilst in eight cases (13.73%) the expression was higher in non-malignant peritumor tissue. Total CD44 expression was higher in tumor tissue compared with paired non-invaded peritumor samples in 47 cases (78.95%). In the remaining cases the difference between paired samples was biologically insignificant. In conclusion, our study suggests that qRT-PCR is feasible in assessing the gene expression profiles of CSCs from CRC, and a promising pathway to be followed for determining how often a person needs screening by colonoscopy and at which age to start. This could improve CRC diagnosis and early patient stratification, and open the way for new oncologic treatment development.