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BackgroundStudies comparing the ability of staining methods to detect eosinophils in formalin‐fixed canine skin are lacking.Hypothesis/ObjectivesThe aim of this study was to compare the effectiveness of eosinophil peroxidase monoclonal antibody (EPXmAb), Luna and haematoxylin and eosin (H&E) to detect eosinophils in fixed canine skin by assessing the following parameters: (i) specificity of eosinophil staining; (ii) extracellular eosinophil granule staining; (iii) tissue background staining; (iv) contrast between eosinophil and surrounding tissue staining; and (v) differences in the number of eosinophils detected by each stain.MethodsThree serial sections of formalin‐fixed, paraffin‐embedded tissues of predominantly eosinophilic skin diseases (n = 8), noneosinophilic skin diseases (n = 7) and normal canine skin (n = 1) were stained with the three stains. Each parameter was independently assessed and scored by two investigators.ResultsLuna and EPXmAb were specific in detecting eosinophils. The EPXmAb was significantly more effective than Luna (P < 0.001) and H&E (P < 0.001) in its ability to detect extracellular eosinophil granules (i.e. to detect intact and released eosinophil granules). The EPXmAb showed significantly less background staining compared with Luna (P = 0.0005). Moreover, significantly more stain contrast was noted with EPXmAb compared with Luna (P = 0.003) and H&E (P < 0.001). There was no significant difference between the mean eosinophil counts among the three stains.Conclusion and clinical importanceThe three stains were shown to be useful to detect and quantify eosinophils in fixed canine skin. The EPXmAb‐based immunohistochemical stain proved to be a novel tool to detect eosinophils in canine skin.