Spermatogenesis in the freshwater pearl mussel Margaritifera laevis was investigated using light and electron microscopy. The testes of M. laevis are composed of numerous acini. We observed type A spermatogonia, large cells of irregular shape, solely near the acinus basal lamina. Type A spermatogonia proliferate and become type B spermatogonia, which are also irregular in shape and form clusters of germ cells of the same developmental stage. The numerous clusters differ with respect to developmental stage and are arranged randomly along the acinus periphery. The central region of the acinus was observed to contain only mature spermatozoa. This germ cell arrangement contrasts that of other bivalvians and may be characteristic of Margaritiferidae and Unionidae. We noted that each germ cell cluster is entirely covered throughout spermatogenesis by Sertoli cells that are loosely bound together. This report is the first to describe the involvement of Sertoli cells in Unionoidea spermatogenesis. Mature spermatozoa of M. laevis are of the primitive sperm type, having a cylindrical head with a discoidal acrosome and a midpiece with five spherical mitochondria.