The establishment of the tetraploid organism is difficult but useful in genetics and breeding. In the present study, we have artificially established an autotetraploid fish line (F₂–F₈) derived from the distant hybridization of Carassius auratus red var. (RR, 2n = 100) (female) × Megalobrama amblycephala (BB, 2n = 48) (male). The autotetraploid line (F₂–F₈) possess four sets of chromosomes from red crucian carp (RRRR, 4n = 200) and produce diploid ova and diploid sperm, which maintains the formation of the autotetraploid line. The F₂ of the autotetraploid fish result from the fertilization of the autodiploidy diploid eggs and diploid sperm from the females and males of F₁ hybrids (RRBB, 4n = 148), which exhibit abnormal chromosome behavior during meiosis as revealed by gynogenesis and backcrossing. This is the first report concerning the establishment of an autotetraploid fish line derived from distant hybridization. The autotetraploid fish line provides an important gamete source for the production of triploids and tetraploids. The autotetraploid fish line also provides an ideal system to investigate the poorly understood mechanisms that drive diploidization in autotetraploids and to study the hybrid progenies' characteristics, including the appearance of new traits that promote a diversity of traits and facilitate adaptation.