Background The unique anatomy of the male reproductive organ reflects its complex function from sperm maturation to their storage for months until emission. Since light microscopy in two dimensions (2d) cannot sufficiently demonstrate its complex morphology, a comprehensive visualization is required to identify pathologic alterations in its entire anatomical context. Objectives Aim of this study was to use three‐dimensional (3d) light sheet fluorescence microscopy (LSFM) to visualize entire murine testes in 3d, label‐free and at subcellular resolution, and to assign local autofluorescence to testicular and deferent structures. Materials and methods Murine testes were fixed with four different fixatives and subsequently cleared with benzoic acid/benzyl benzoate. Hereafter, complete murine testes were scanned with LSFM with different fluorescence filter sets and subsequently embedded in paraffin for further conventional planar histology. Results Autofluorescence signals of the murine reproductive organ allowed the unambiguous identification of the testicular anatomy from the seminiferous tubules to the vas deferens with their specific stratification independent of the used fixative. Blood vessels were visualized from the pampiniform plexus to the small capillaries of single tubules. Moreover, due to the specific intrinsic fluorescence properties of the efferent ducts and the epididymis, luminal caliber, the epithelial stratification and retronuclear cytoplasmic inclusions gave a unique insight into the interface of both morphological structures. Subsequent 2d histology confirmed the identified morphological structures. Discussion LSFM analysis of the murine reproductive organ allows due to its intrinsic fluorescence a simple, label‐free 3d assessment of its entire duct morphology, the epithelial composition, and the associated blood supply in its anatomical relation. Conclusion LSFM provides the technical basis for comprehensive analyses of pathologically altered murine testes in its entirety by depicting specific autofluorescence. Thereby it facilitates mouse studies of testicular disease or their drug‐related alterations in more detail potentially for clinical translation assessing human testicular biopsies.