Heat-stable protein fraction in seeds is believed to enrich many proteins functioning in the acquisition of stress-tolerance of seeds. In this study, the composition of heat-stable protein fraction in imbibed cowpea (Vigna unguiculata) seeds was analyzed by SDS-PAGE and twodimensional gel electrophoresis coupled with mass spectrometry. The results indicated that approximately 12.4 % of seed soluble proteins were stable after heat treatment at 100 C for 10 min. Twenty-two putative heat-stable proteins were identified using MALDI-TOF/TOF MS. Most of these heat-stable proteins were late embryogenesis abundant proteins, and there were other stress-related proteins including Cu/Zn superoxide dismutase and 17.4 kDa Class I heat-shock protein. A cyclophilin protein, a cleavage and polyadenylation specificity factor and a Pumilio-family RNA binding protein were also present in the heat-stable fraction. The identified heat-stable proteins were more hydrophilic proteins and may accumulate to stabilize cellular components and maintain seed viability during seed development and germination.