Due to its rarity, it takes months for patients to be correctly diagnosed with pemphigus vulgaris. This delay can be exacerbated by factors such as incorrect biopsy site selection. When the epidermis detaches from the dermis, evaluating fluorescence patterns is impossible. This monocentric, retrospective study aimed to assess the clinical significance of the honeycomb-like staining pattern of adnexal structures in patients with pemphigus vulgaris. The study was conducted in Munich, from 1 January 2012 to 31 December 2020. Forty-four biopsy samples for direct immunofluorescence microscopy from pemphigus patients along with 44 samples from control patients were included. The fluorescence intensity of adnexal structures did not differ significantly from that of the epidermis in pemphigus, except for hair follicles and sweat glands with C3 staining, where the epidermis showed stronger fluorescence. The sensitivity for the characteristic fluorescence pattern of at least 1 adnexal structure was 88.64% (n = 39/44, 95% CI 75.44% to 96.21%), with 100% specificity. The positive predictive value of adnexal fluorescence was 100%, and the negative predictive value was 89.8%. It was found that the honeycomb-like staining pattern of sweat glands, sweat gland ducts, and hair follicles is a highly specific marker for pemphigus vulgaris.