For the first time, DNA content of the sperm of tetraploid mud loach (Misgurnus mizolepis) males and the ploidy status of progenies generated by crossing tetraploid males with diploid females are described. Of 48 tetraploid males tested, 12 showed significantly retarded gonad development, but the other 36 produced functional sperm. Of these 36, 26 produced haploid sperm which, on fertilizing haploid eggs, generated diploid progenies. Seven tetraploid males were mosaics in their sperm, as indicated by the production of diploid, aneuploid and/or triploid offspring. Only three males produced diploid sperm rendering the production of all-triploid progenies. The DNA content of sperm of tetraploid males was consistent throughout three progeny tests, i.e., the haploid sperm-producing 4n males persisted to produce haploid sperm only; likewise, the diploid sperm producing 4n males consistently produced diploid sperm only, when progeny testing was extended to 3 successive but alternate years. Hence, a careful and direct examination of the DNA profile of sperm from tetraploid males is a pre-requisite for reproductive containment of genetically modified fish.