Introduction The cause of penile curvature (PC) in hypospadias remains unclear. Surgical correction through dartos fascia resection can straighten the penis, suggesting its role in superficial PC. This study aimed to compare collagen density and gene expression of COL1A1, COL3A1, and ELN in proximal and distal dartos fascia of hypospadias patients and age-matched controls. Methods In this prospective case–control study, 50 hypospadias patients (25 distal, 25 proximal) and 50 age-matched controls were included. Dartos samples were collected from proximal and distal ventral penile regions in hypospadias cases, and from the coronal area in controls. Collagen density was assessed histologically using Masson's trichrome staining, and gene expression was quantified using real-time polymerase chain reaction (RT-PCR). Subgroup analyses were performed based on hypospadias type and PC severity. Results Significant differences of collagen density, COL1A1, COL3A1, and ELN were observed among distal dartos, proximal dartos, and controls (p < 0.01). The proximal dartos exhibited the most consistent reduction across all markers (p < 0.01). In distal hypospadias and mild PC, the distal dartos remained comparable to controls (p > 0.05), while the proximal dartos showed significantly reduced values (p < 0.001). In proximal hypospadias and severe PC, both dartos regions were affected, with the distal dartos showing more pronounced reductions in COL3A1 and ELN (p < 0.05). The COL3A1:COL1A1 ratio was also significantly reduced in hypospadias patients, particularly in the proximal dartos and in those with moderate to severe penile curvature. Conclusion The extracellular matrix composition of dartos fascia in hypospadias varies according to anatomical location and severity. The distal dartos may be preserved for neourethral coverage in distal hypospadias and mild PC, while excision may be more appropriate in proximal hypospadias (Level of Evidence: III). © 2025 The Author(s).