Injury caused by imbibitional chilling is a common phenomenon during rehydration of desiccated seeds, and usually causes seriously compromises seedling emergence and crop yield. Restoration of the cell membranes is a critical event during imbibition, moreover it is very important in responses to water and temperature stress. However, the changes in membrane structure during seed imbibition, especially those related to membrane fluidity, have yet to be investigated. This study compared changes in the level of unsaturation of membrane lipids (doublebond index, DBI) between chillingtolerant ‘LX’ and chillingsensitive ‘R5’ soybean cultivars during imbibitional chilling. After imbibition at normal temperature (25℃), seeds of LX and R5 showed similar changes in the level of lipid unsaturation, with increased DBI values for plastidic lipids and reduced levels of extraplastidic phosphatidylglycerol (PG). In contrast, there were dramatic differences in the changes of DBI between LX and R5 following imbibition under chilling conditions (4℃). Chilling only delayed the increase of plastidic DBI in seeds of the tolerant cultivar LX, whereas plastidic DBI did not increase or even decreased in the sensitive cultivar R5 under these conditions. Priming of seeds by incubation in 33% polyethylene glycol (PEG) did not directly cause changes in DBI, however, the subtle but significant changes in DBI that it induced prepared the soybean strain for germination. Plastidic DBI increased during phases II and III of germination in PEGosmoconditioned R5 seeds under imbibitional chilling stress; this increase was similar to the patterns of increase in DBI of LX. Our results suggest that chilled imbibition delays or prevents the increase of the degree of unsaturation in plastidic membrane lipids, that tolerance to imbibitional chilling in soybean is positively associated with an increase in the level of unsaturation of plastidic membrane lipids, and that increased plastidic DBI could improve tolerance of imbibitional chilling.