Abstract: Poly (A)⁺ RNA were purified using Oligotex mRNA Kits (Qiagen) from peripheral blood leukocytes of 20 Holstein cows with mastitis and 20 health Holstein cows in lactation as control; single- and double-stranded cDNA were synthesized from the poly(A)+ RNA using PCR-Select^TM cDNA Subtraction Kit (Clontech) and further digested using RsaⅠ into cDNA fragments sized from 400 to 600 bp (dscDNA); dscDNAs from the mastitis cows (as tester) were divided into two portions which were ligated separately with a different cDNA adaptor; the ligated cDNA were hybridized twice with the dscDNA of the healthy cows at 68 ℃ for 8 h each; the products after double hybridizations were diluted by 200 times and then used for suppression PCR twice; the secondary PCR products was inserted into PGEM-T vector and transformed into E.coli TOP10 competent cells; 610 positive clones were obtained; identification of the inserted cDNA fragments in subtractive library was done using PCR. The results showed that there were inserted fragments sized by 250~750 bp in the 16 randomly selected positive clones, which would provide useful baseline for screening and cloning specific mastitis resistant genes and understanding the molecular mechanism of mastitis resistance in dairy cow.