Abstract Introduction Colorectal cancer (CRC) stem cells or tumor-initiating cells (Co-TIC) are implicated in both cancer recurrence and extranodal metastasis. CD133 and CXCR4 are specific cell surface markers that are indicators of Co-TIC. The presence of lymph node (LN) metastases is one of the strongest negative prognostic factors for CRC patients. We examined the relationship between the Co-TIC markers CD133 and CXCR4 and LN involvement in CRC. Methods CRC cells were isolated via enzymatic digestion. CD133+, CXCR4+, and double-positive CRC cells were detected by fluorescence-activated cell sorting analysis. The percentages of CD133+, CXCR4+, and double-positive cells were identified and correlated to the number and percentage of positive LN on staging. Results Twenty-seven samples underwent fluorescence-activated cell sorting analysis. The mean percentage of CD133+ cells was 3.94% (range 0.15%–19.06%). The mean percentage of CXCR4+ cells was 6.15% (range 0%–27.11%). The mean percentage of CD133+CXCR4+ cells was 0.45% (range 0%–2.08%). Thirteen patients had LN metastasis: 8 N1 disease and 5 N2 disease. The correlation coefficients between the percentage of Co-TIC marker–positive cells and percentage of positive LN were r = 0.58 (P = 0.0016) for CD133+ cells, r = 0.36 (P = 0.5868) for CXCR4+ cells, and r = 0.56 (P = 0.0022) for double-positive cells. Discussion Our results show CD133+ and CD133+CXCR4+ cancer cells correlate with the presence of LN metastasis in CRC. Further studies will examine whether these markers can give consistent prognostic information and may help to develop novel diagnostic and therapeutic options.