The etiology of primary pilonidal sinus disease (PSD) remains unclear. Current understanding suggests that sharp hair fragments from the occiput contribute to the formation of PSD. In 2009, Akinci et al. reported a correlation between PSD and a deeper natal cleft. We investigated the association between intergluteal fold (IGF) depth and PSD risk using a standardized five-step measuring protocol. Our study included 95 PSD patients and 105 non-PSD individuals, and measurements were taken from the glabella sacralis to the anus in a controlled in-house setting after obtaining informed consent from voluntary participants of a northern German population. The mean (± SD) intergluteal depth progressively increased from the intergluteal opening at the glabella sacralis at 9.1 (± 3.4) mm to a maximum of 62.6 (± 10.4) mm. Notably the deepest point was consistently observed at the anus, where PSD occurence is rare. No significant difference in intergluteal fold depth between PSD and non-PSD patients was found. Additionally, PSD predominantly developed in the proximal (cranial) third of the intergluteal fold, despite the maximum depth being in the distal region. These findings suggest that intergluteal fold depth is not a risk factor for PSD.