Abstract:microRNAs(miRNAs) is a class of single-strand small RNAs that regulate gene expression by binding to complementary sequences in the 3' untranslated region(3'UTR) of target mRNAs. In a sense, identification of miRNAs and their targets in mammary gland tissue is essential to proclaim the function of miRNAs on gene expression and regulation. In the present study, small RNAs were extracted from porcine mammary gland tissue of Jinhua and Yorkshire breed respectively and used to construct a cDNA library. Then it was sequenced by Illumina Genome AnalyzerⅡx. Mappable sequences were obtained from raw sequences which filtered junk, short, simple, low copy and certain known types of RNA sequences. BLASTNs were performed between miRNABase Genome, EST database and mappable sequences. Different miRNAs detected between two pig breeds were conducted , and top ten abundance of miRNAs were selected for further bioinformatics analysis. The results showed that the reads of mappable sequence was 9 672 024 in the mammary gland tissue of Jinhua pig and 6 946 905 in Yorkshire pig, while the number of unique miRNA detected in Jinhua pig was 848 and 683 in Yorkshire pig. Moreover, 369 and 231potential novel unique miRNAs in Jinhua and Yorkshire pigs were discovered, respectively. Totally 288 miRNAs had significant difference between Jinhua and Yorkshire pigs(P<0.01), and 1 829 targets in accordance with top ten abundance of miRNAs were also revealed. These targets were primarily involved in many pathways such as antigen processing and presentation, typeⅠdiabetes mellitus, gap junction, allograft rejection and so on, suggest that these miRNAs have widely roles in physiological metabolism processing of mammary gland. Our study enriches the porcine miRNABase and provides a basis for researching the function and the regulated mechanism of miRNA in mammary gland tissue.