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Aims: Intraoperative consultation using frozen sections (FSs) is an integral component of clinical practice. As a quality control measure, FS diagnosis is subsequently compared with the findings on a FS control slide. These control slides can be used for immunohistochemistry, and the immunohistochemical performance in FS controls is known to be antibody‐dependent. Isocitrate dehydrogenase (IDH) is mutated in >80% of lower‐grade infiltrating gliomas in adults and in ~10% of glioblastomas, with IDH1 (R132H) being the most common mutation. IDH status is used as a major classifier of glioma. An IDH1 (R132H)‐specific antibody (H09) has been accepted as a robust surrogate for genetic testing. In this study, we aimed to determine how previous freezing and thawing affects IDH1 immunohistochemistry. Methods and results: Thirty cases of IDH1 (R132H)‐mutant diffuse glioma, which were originally assessed on FSs, were retrieved. The positive IDH1 (R132H) status of each case was previously determined with pyrosequencing and H09 immunohistochemistry on permanent sections. The FS control tissue of each case was immunostained with H09 antibody. Among 30 gliomas, 25 showed negative reactivity on FS control slides, whereas, in the remaining five, the staining was uninterpretable, owing to a high diffuse background. Of the former 25 specimens, 20 showed at least focal areas with variably increased levels of background staining, whereas the remaining five specimens showed a relatively pristine background. Conclusions: We conclude that IDH1 (R132H)‐specific (H09) immunohistochemistry often results in false‐negative reactions on FS controls with focally increased background staining, and this application should be avoided in clinical practice. [ABSTRACT FROM AUTHOR]