<p>Although the cyprinid zebrafish has been widely used to study gametogenesis and gonadal differentiation, it has remarkable limitations, such as the loss of sex determinants in laboratory strains, a juvenile ovary stage in both sexes, and oocyte defects generally leading to sex reversal. Here, we develop a novel small cyprinid model for studying gonadal differentiation and gametogenesis, Juji (<i>Gobiocypris rarus</i>), which possesses a genetic sex determination system. We generate a transgenic line of Juji, <i>Tg(ddx4:EGFP-UTRddx4)</i>, enabling lifetime visualization of germline development, and find that the dimorphic expression of <i>ddx4:EGFP</i>, rather than primordial germ cell number, at early stages determines the sexual fate of adults. RNA-seq analysis of juvenile ovaries and testes prior to germ cell differentiation identifies sexually dimorphic genes and signaling pathways. Several key factors, such as <i>foxl2l</i> and <i>dmrt1</i>, which drive gonadal differentiation, are among the sexually dimorphic genes. Knockout of <i>dmrt1</i> leads to sex reversal in juvenile males, with all mutants developing into females. Furthermore, transplanting germline stem cells from genetic males into <i>dmrt1</i> mutants results in all transplants developing into females, highlighting the essential Sertoli-cell-specific role of <i>dmrt1</i> in triggering spermatogenesis. Our study establishes Juji as a promising model for investigating sex differentiation, gametogenesis, and gonadal development.</p>