Circulating tumor cells (CTC) in blood have attracted attention both as potential seeds for metastasis and as biomarkers. However, most CTC detection systems might miss epithelial-mesenchymal transition (EMT)-induced metastatic cells because detection is based on epithelial markers. First, to discover novel markers capable of detecting CTCs in which EMT has not been repressed, microarray analysis of 132 colorectal cancers (CRC) from Japanese patients was conducted, and 2,969 genes were detected that were overexpressed relative to normal colon mucosa. From the detected genes, we selected those that were overexpressed CRC with distant metastasis. Then, we analyzed the CRC metastasis-specific genes (n ¼ 22) to determine whether they were expressed in normal circulation. As a result, PLS3 was discovered as a CTC marker that was expressed in metastatic CRC cells but not in normal circulation. Using fluorescent immunocytochemistry, we validated that PLS3 was expressed in EMTinduced CTC in peripheral blood from patients with CRC with distant metastasis. PLS3-expressing cells were detected in the peripheral blood of approximately one-third of an independent set of 711 Japanese patients with CRC. Multivariate analysis showed that PLS3-positive CTC was independently associated with prognosis in the training set (n ¼ 381) and the validation set [n ¼ 330; HR ¼ 2.17; 95% confidence interval (CI) ¼ 1.38-3.40 and HR ¼ 3.92; 95% CI ¼ 2.27-6.85]. The association between PLS3-positive CTC and prognosis was particularly strong in patients with Dukes B (HR ¼ 4.07; 95% CI ¼ 1.50-11.57) and Dukes C (HR ¼ 2.57; 95% CI ¼ 1.42-4.63). PLS3 is a novel marker for metastatic CRC cells, and it possesses significant prognostic value. Cancer Res; 73(7); 2059-69. Ó2012 AACR.