Bloch) were dealt with ENU (1-ethyl-l-nitrosourea) solution (92.8mg/L) for about two weeks, then restored in fresh water for at least another two weeks. Then single mating was conducted to produce F₁ siblings. From AFLP fingerprint, I type(deletion) specific site and II type (insertion) specific site were detected from genomic DNA (gDNA) of 55 F₁ individuals, among which 30 from mutation group and 25 from control group. The whole mitochondrial DNA (mtDNA) sequences were compared between six F₁ individuals ♀ from its counterparts. Compared with the control group, the mutative loci could be found in the entire mtDNA in mutation group which mainly located in 13 protein-encoding genes,12SrRNA gene, 16S rRNA gene and D-loop control regions. The frequencies of ENU mutation could be up to 38% in calculating nucleotide changes of NADH1 and NADH2 sequences alone. The mutated F₁ individuals have similar mutative locations and loci based on the results of AFLP and mtDNA sequence alignment, with the mutated F₁ males seeming to have obtained more mutative loci than those of F₁ females. This study offers direct data for the estimation of ENU efficiency in gibel carps, and provides a new trial for fish breeding.