In this study,using the methylation sensitive amplified polymorphism(MSAP)method,we globally assessed the effect of low temperature adaptation on the level and variations of cytosine methylation alterations at CCGG sites of genomic DNA in Nile tilapia(Oreochromis niloticus)strains with good cold-resistance characters developed by the successive and directional selection breeding of multi-generations.Total of 849 fragments were identified by 18 pairs of se1ective primer combinations of which 411 and 438 were detected in cold-resistance tilapia strain and the control,respectively.Among these fragments,the methylation sites were 72 and 104,and the corresponding total-methylation levels were at 17.52% and 23.74%,respectively.The full methylation sites at the internal Cs were 37 and 65,and the corresponding full methylation levels were at 9.00% and 14.84%,respectively,and the hemi methylation sites at the external Cs were 35 and 39,and the corresponding hemi-methylation levels were at 8.52% and 8.90% in cold-resistance tilapia strain and the control,respectively.Further analysis of DNA methylation indicated that a statistically decrease in the overall level of total-methylation levels and both methylation types was detected in cold-resistance tilapia strain compared with the control(decrease at 6.22%,5.84% and 0.38%,respectively),and the changes of DNA methylation pattern were recognized mainly in the full methylation sites.The level of decrease in DNA methylation suggested that the levels of genomic DNA methylation were changed during the successive cold stress of multi-generations in tilapia,and it also indicated that DNA methylation alteration in tilapia treated with cold stress was mainly through de-methylation that occurred in some CCGG sites.All these results implied that the change of DNA methylation was closely associated with cold tolerance of tilapia.The present findings are valuable to further explore the application potential of DNA methylation alteration in tilapia genetic improvement and provide a new method and theoretical basis for fish stress resistance breeding.