We utilized the high‐throughput tissue microarray method to characterize immunohistochemical expression patterns with correlations to prognosis in rectal cancer. Immunostaining for the markers Ki‐67, Bcl‐2, p53, EGFR, E‐cadherin, β‐catenin, MLH1 and MSH2 was performed in 269 rectal cancers. Expression profiles were correlated to metastasis‐free survival. Immunostaining revealed frequent upregulation and/or aberrant staining patterns for several of the markers, but Ki‐67, p53, Bcl‐2 and EGFR did not show any correlation to prognosis. However, reduced membranous staining for β‐catenin (p = 0.04), lack of cytoplasmic staining for β‐catenin (p = 0.04), reduced membranous staining for E‐cadherin (p = 0.02) and lack of cytoplasmic staining for E‐cadherin (p = 0.02) correlated with metastatic disease. Multivariate analysis including the factors Dukes' stage and tumor differentiation grade demonstrated increased risk of metastatic disease in tumors with lack of cytoplasmic staining for β‐catenin (HR = 3.1, p = 0.02), reduced membranous staining for β‐catenin (HR = 1.7, p = 0.06) and reduced membranous staining for E‐cadherin (HR = 2.1, p = 0.06). Loss of MMR protein expression was confirmed to be a rare event in rectal cancer with loss of MLH1 staining in 3% and MSH2 in 1% of the tumors. The lack of prognostic information contributed by most of these markers suggests that single markers for prognosis may be of limited value in rectal cancer. However, altered expression of β‐catenin and E‐cadherin correlated with metastatic disease, and these markers may have prognostic importance in rectal cancer. © 2004 Wiley‐Liss, Inc.