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The highly conserved transcription factor Pax6 is involved in the development of the eyes, brain, and pancreas in vertebrates and invertebrates, whereas the additional expression pattern in other organs is still elusive. In this study, we cloned and characterized two <i>pax6</i> homologs in blunt snout bream (<i>Megalobrama amblycephala</i>), named <i>Mapax6a</i> and <i>Mapax6b</i>. The protein alignment and phylogenetic tree showed that Mapax6a and Mapax6b were highly conserved compared with their counterparts in other species. Genomic information analysis revealed that the synteny conservation of Wilms tumor, Aniridia, genitourinary abnormalities, and mental retardation loci was also maintained in this species. By reverse transcription polymerase chain reaction, the expression of <i>Mapax6a</i> was later than that of <i>Mapax6b</i> which was found in the blastula stage, while the expression of <i>Mapax6a</i> started from the somite stage, and both of them persisted in a subsequent stage during the embryonic development. By RNA and protein detection, <i>Mapax6a</i> and <i>Mapax6b</i> were detected in the eye and brain as canonic patterns, and most importantly, they were also enriched in germ cells of the testis and ovary. Therefore, our findings validate the duplication of <i>pax6</i> in fish, confirm the classical expression patterns in the brain and eye, and, for the first time, present a new acquisition of <i>Mapax6a</i> and <i>Mapax6b</i> in gonadal germ cells in particular. Therefore, our results enrich the expression pattern and evolutionary relationship of <i>pax6</i> by suggesting that duplicated <i>Mapax6</i> is involved in gametogenesis in <i>Megalobrama amblycephala</i>.