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Introduction There have been problems with low qualification operator-related complications and failures of transurethral seminal vesiculoscopy (TSV) in China. Aim To study the guiding role of seminal tract anatomical study (STAS) in TSV. Material and methods We performed STAS to study the structure, morphology, duct trajectory, and anatomical relationships between the seminal vesicles and the adjacent tissue in pelvic specimens from 12 adult cadavers. Then the surgical effects and complications of 82 cases of TSV performed by 3 doctors were retrospectively studied to compare the difference between the two groups of before and after the anatomical study. Results The anatomical studies of the 12 adult cadaveric pelvis specimens identified the lengths and widths of the right- and left-side seminal vesicles and tracts. The TSV can treat lesions located in the distal seminal tract and vesicle, but proximal lesions cannot be reached, which is an anatomical limitation of this technique. There were significant differences in the surgical times and the surgical validity rates between the 2 groups. Conclusions Our anatomical study of the seminal tract and seminal vesicles is valuable for guiding TSV in clinical practice.