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The purpose of this study was to tendency the expression of microRNA and xenomiR in hay of two alfalfa varieties (cv.Zhongmu No.1, cv.Xinyan No.52), and analyze the expression level of miR-168c, miR-156a and miR-166e of alfalfa in cow serum, milk and colostrum. The miRs in fresh and dry samples of two alfalfa varieties were sequenced by high-throughput RNA sequencing technology, RT-qPCR was used to analyze the expression levels of miR-156a, miR-166e and miR-168c. In addition, the target gene of miR168c in dairy cows was verified by Double-Luciferase Report.The results showed that: (1) 771 miRs were detected, 113 and 84 miRs expression levels in dry samples were up- and down-regulated, 8855 target genes were predicted. (2) miR-156a, miR-166e and miR-168c expression levels in dry samples of two alfalfa varieties were opposite, which were down-regulated in cv.Zhongmu 1 and up-regulated in cv.Xinyan 52. (3) The expression levels of miR-168c, miR-156a and miR-166e in high producing dairy cow serum were significantly higher than low producing dairy cow (p |