Soybean is planted worldwide and its productivity is significantly hampered by salinity. Development of salt tolerant cultivars is necessary for promoting soybean production. Despite wealth of information generated on salt tolerance mechanism, its basics still remain elusive. A continued effort is needed to understand the salt tolerance mechanism in soybean using suitable molecular tools. To better understand the molecular basis of the responses of soybean to salt stress and to get an enrichment of critical salt stress responsive genes in soybean, suppression subtractive hybridization libraries (SSH) are constructed for the root tissue of two cultivated soybean genotypes, one was tolerant and the other was sensitive to salt stress. To compare the responses of plants in salt treatment and non-treatment, SSH1 was constructed for the salt-tolerant cultivar Wenfeng 7 and SSH2 was constructed for the salt-sensitive cultivar Union. From the two SSH cDNA libraries, a total of 379 high quality ESTs were obtained. These ESTs were then annotated by performing sequence similarity searches against the NCBI nr (National Center for Biotechnology Information protein non-redundant) database using the BLASTX program. Sixty-three genes from SSH1 and 49 genes from SSH2 could be assigned putative function. On the other hand, 25 ESTs of SSH1 which may be not the salt tolerance-related genes were removed by comparing and analyzing the ESTs from the two SSH libraries, which increased the proportion of the genes related to salt tolerance in SSH1. These results suggested that the novel way could realize low background of SSH and high level enrichment of target cDNAs to some extent. Soybean is planted worldwide and its productivity is significantly hampered by salinity. Development of salt tolerant cultivars is necessary for promoting soybean production. Despite continuing the development of salt tolerance mechanism, its basics still remain elusive. A continued effort is needed to understand the salt tolerance mechanism in soybean using suitable molecular tools. To better understand the molecular basis of the responses of soybean to salt stress and to obtain an enrichment of critical salt stress responsive genes in soybean, suppression subtractive hybridization libraries (SSH) are constructed for the root tissue of two cultivated soybean genotypes, one was tolerant and the other was sensitive to salt stress. To compare the responses of plants in salt treatment and non-treatment, SSH1 was constructed for the salt-tolerant cultivar Wenfeng 7 and SSH2 was constructed for the salt -sensitive cultivar Union. From the two SSH cDNA libraries, a total of 379 high quality The ESTs were then annotated by performing sequence similarity searches against the NCBI nr (National Center for Biotechnology Information protein non-redundant) database using the BLASTX program. Sixty-three genes from SSH1 and 49 genes from SSH2 could be assigned putative On the other hand, 25 ESTs of SSH1 which may be not the salt tolerance-related genes were removed by comparing and analyzing the ESTs from the two SSH libraries, which increased the proportion of the genes related to salt tolerance in SSH1. These results suggested that the novel way could realize low background of SSH and high level enrichment of target cDNAs to some extent.