Objective: The purpose of this study is to investigate whether there was a difference between the midline skin and the healthy skin in the lateral by means of total amount of collagen and Type I/III ratio which was the indicator of the collagen structure. Material and methods: Fifty patients with pilonidal sinus disease were enrolled. Samples were prepared from the midline skin of the sinus where the holes were located and lateral skin of the resected material. Results: It was determined that the lateral line had significantly more collagen intensity and a higher collagen Type I/III ratio (p < 0.001). Conclusions: One of the reasons why hair mostly pricks into the midline in the intergluteal sulcus in pilonidal sinus disease is the fact that the amount of total collagen and collagen Type I/III ratio of the midline are lower than those of the lateral tissue. Complications are more common in cases with low Type I/III ratio and low total collagen rates.