Colorectal carcinoma shows several sex‐related differences with regard to incidence, response to chemotherapy and microsatellite instability. These differences may relate to differential expression of ERβ1 (wild‐type) as well as the truncated ERβ2 and ERβ5 splice variant isoforms, which have recently been detected in normal and malignant colorectal epithelium. This hypothesis was tested through the study of ERβ isoform protein and/or mRNA expression amongst 91 primary colorectal carcinoma cases and 20 colorectal carcinoma cell lines. Study of the latter showed an absolute correlation between mRNA and protein expressions for ERβ1 and ERβ2. ERβ1 and ERβ2 protein expression was lost in 22% and 49%, respectively, of the primary colorectal carcinomas. By contrast, ERβ5 expression was found in all primary colorectal carcinomas and all colorectal carcinoma cell lines studied. Lower ERβ1 protein expression was associated with poorer differentiation, higher pT stage and absence of microsatellite instability. Higher ERβ2 protein expression was associated with right‐sided location and presence of lymph node metastases. Protein expression of ERβ1 correlated positively with expression of the oestrogen‐responsive protein trefoil factor 1 (TFF1). There was no correlation between ERβ protein isoform expression and response to 5‐fluorouracil therapy, tumour proliferation, or thymidylate synthase expression. These data suggest that ERβ1 and/or ERβ2 isoform expression may have prognostic value and may explain sex‐related differences in microsatellite instability and colorectal carcinoma. The opposing associations shown by ERβ1 and/or ERβ2 in relation to colorectal carcinoma are in keeping with differential activities shown by the two isoforms. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.