Acantholytic dermatophytosis is a rarely reported condition of dogs that clinically and histopathologically mimics superficial pemphigus (erythematosus, foliaceus). Histologically, periodic acid-Schiff (PAS) and Grocott's methenamine-silver (GMS) are often necessary to show the fungus. A retrospective histopathological study was conducted on 190 canine skin biopsy specimens: 95 each with the diagnosis of canine dermatophytosis or of superficial pemphigus. All specimens were stained with haematoxylin and eosin, PAS, and GMS. Dermatophytes were not seen in any superficial pemphigus cases. Acantholytic keratinocytes were noted in 14% of the dermatophytosis cases, none of which had clinical signs consistent with superficial pemphigus. Among cases with acantholytic keratinocytes, superficial pemphigus had significantly more acantholytic cells than dermatophytosis (P = 0.02). When comparing face and nonface cases, there was no difference in prevalence of acantholytic keratinocytes in dermatophytosis or number of acantholytic keratinocytes in superficial pemphigus. All dermatophyte cases were both GMS and PAS positive with neither stain being visually superior. No dermatophyte cases where acantholytic keratinocytes were noted had a history, clinical signs and histopathological features compatible with acantholytic dermatophytosis.