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The Wnt/β-catenin signaling pathway is dysregulated in different types of neoplasms including colorectal cancer (CRC). Aberrant activation of this signaling pathway is a key early event in the development of colorectal neoplasms, and is mainly caused by loss of function mutations in Adenomatous Polyposis Coli (APC), and less frequently by β-catenin stabilization mutations via missense or interstitial genomic deletions in CTNNB1. In this study, we have defined an immunohistochemical algorithm to dissect Wnt pathway alterations in formalin-fixed and paraffin-embedded neoplastic tissues. Basically, consecutive sections of tumor specimens were stained by immunohistochemistry with two different monoclonal antibodies against β-catenin: one (anti-active β-catenin antibody) recognizes hypo-phosphorylated β-catenin and the other recognizes the total pool of β-catenin. We validated the strategy in the HCT116 CRC cell line which has an in-frame deletion of β-catenin serine 45, and then studied human tumor microarrays containing colon adenomas, CRCs, solid pseudopapillary neoplasms of the pancreas as well as the whole tissue sections of CRCs, desmoid fibromatosis, and pilomatrixoma of the skin. In some tumors, we found strong β-catenin cytoplasmic and/or nuclear staining with the total β-catenin antibody but no staining with the anti-active β-catenin antibody. This was inferred to be an altered/mutant β-catenin staining pattern. All six colon adenomas of the 126 total adenomas studied for the altered/mutant β-catenin staining pattern had presumptively pathogenic point mutations or deletions in CTNNB1. Four of 10 CRCs with the alterated/mutant β-catenin staining pattern studied in depth, from 181 total CRCs from tissue microarray, had pathogenic CTNNB1 mutations. The frequencies of CTNNB1 alterations in non-colonic tumors with altered/mutant β-catenin staining ranged between 46 and 100%. Our results demonstrate that the immunohistochemical approach described here can detect oncogenic forms of β-catenin in primary tissue samples and can also highlight other tumors with presumptive novel defects activating the Wnt/β-catenin pathway.