Spermatogenesis in teleosts is essential for reproductive function; however, it varies considerably among species. The testis of the viviparous molly fish (Poecilia sphenops) was examined using both ultrastructural and immunohistochemical methods. The testis displays a restricted lobular type, where germ cells develop synchronously within Sertoli cell-forming cysts. Transmission electron microscopy (TEM) revealed all stages of spermatogenesis. Mature sperm are at the apex of the cysts and migrate toward the sperm ducts. Sperm duct epithelium is lined by cuboidal cells joined by tight junctions, with apical cilia and desmosomal complexes contributing to transport and structural integrity. The sperm ducts showed strong Periodic Acid-Schiff (PAS)-positive expression among negative stained spermatocysts. Centrally, a cavity serves as a storage area for spermatozoa that are organized into unencapsulated bundles known as spermatozeugmata. Sertoli cells exhibited extended cytoplasmic processes that supported developing germ cells, whereas Leydig cells occupied the interstitial tissue, contributing to hormonal regulation. Immunohistochemical labeling demonstrated strong vimentin expression in Sertoli cells and telocytes, indicating their mesenchymal origin and structural role. Calretinin expression was confined to Leydig cells and certain ductal epithelial cells, supporting its use as a marker for steroidogenic and secretory functions. These findings provide new insights into the testicular specialization of P. sphenops, highlighting key somatic-germ cell interactions, ductal adaptations, and marker expression patterns that underlie male reproductive success in viviparous fish.