Among wild loach (Misgurnus anguillicaudatus) in China, natural tetraploid individuals with 4n = 100 chromosomes appear with sympatric diploid individuals with 2n = 50 chromosomes and low frequencies of natural triploid individuals are also found. However, Chinese polyploid loach specimens have not yet been examined for fertility and ploidy of gametes. Here, we found almost the same concentration per unit volume (cells ml−1), duration of active progressive motility (s) and rate of motile spermatozoa after activation (%) in sperm taken from tetraploid males when compared with sperm from diploid males. Tetraploid females normally laid fertile diploid eggs with larger diameter in size and tetraploid males generated functional diploid sperm. Thus, viable triploid progeny with 3n = 75 chromosomes occurred from crosses between diploid and tetraploid loaches. Natural triploid females laid fertile haploid eggs, but the male produced aneuploid (1.2n–2.2n) spermatozoa causing the decrease of viabilities of the resultant progeny.