Abstract: The objective of this study was to use suppression subtractive hybridization (SSH) to screen the differentially expressed genes in mammary gland of Xinong Saanen Dairy goats at the middle and late lactation stages, and the expression abundance of several genes were analyzed by realtime quantitative PCR (QPCR) to explore the gene expression mechanism of mammary gland in the different lactation stages of dairy goats. The subtracted cDNA libraries (ML and LM) were constructed by using mRNA of mammary gland of Xinong Saanen Dairy goat at the middle and late lactation stages as the Tester and/or Driver, clones were randomly selected and sequenced for sequence alignment, the expression abundance of several genes in mammary gland was analyzed. 30 randomly selected clones from the constructed cDNA subtraction libraries of mammary gland at middle and late lactation stages were sequenced, and the differentially expressed genes related with physiological process including cell apoptosis, antioxidation, lipid and energy metabolism etc. were obtained. Five positive clones were observed from 6 genes assayed by quantitative PCR, the expression levels of which were 1.35.5 folds higher in mammary gland of either middle or late lactation stage. The results showed that the forward and reverse subtracted cDNA libraries of mammary gland at middle and late lactation stages were constructed using SSH, the screening of 24 differentially expressed genes laid the foundation for the further studies on gene regulation in the mammary gland of dairy goats.