In order to definite the effect of salt pretreatment on barley seed germination and microspore culture, the salt tolerant cultivar ‘H11’ and salt sensitive cultivar ‘H30’ were used as materials and the germination rate, taproot length during the seed germination as well as the callus yield in the microspore culture stage were compared under the different salt pretreatment conditions. Meanwhile, the expression of four key genes to salt stress at the microspore stage were also investigated in the two cultivars. The results showed that the seeds and microspores of ‘H30’ were less tolerant to salt than ‘H11’ under 0 g/L NaCl and low NaCl pretreatment conditions, but under the high concentration of NaCl pretreatment (15 g/L NaCl in germination stage and 300 mg/L in microspore stage), the salt tolerance of ‘H30’ was significantly enhanced, and the germination rate, taproot length as well as callus yield with high concentration of salt stress were significantly superior to that of ‘H11’. Through the correlation analysis of germination rate, taproot length and callus yield between the two cultivars, it was found that the relative values of the differences between the two cultivars at the germination stage and the microspore stage were significantly positively correlated. Moreover, in the microspore culture stage, after 300 mg/L NaCl pretreatment, the genes HvsHSP1 and HveIF1A of ‘H30’ were significantly up-regulated in comparison with ‘H11’ under higher concentration of salt stress. It is concluded that the differences in the response of tested barley cultivars to salt pretreatment during seed germination and microspore stage may be consistent. HvsHSP1and HveIF1A of salt sensitive cultivar may be related with the response of NaCl induction in the microspore stage.