Following the concept of whole organism, we have extracted total protein from the Bombyx mori for the identification and analysis of HSPs. Expression of 90 kDa HSP in first, second and third instars, 84 kDa in fourth instar and 90-, 84-, 62-, 60-, 52- and 33-kDa HSPs in fifth instar larvae of tropical polyvoltine and bivoltine silkworm strains were obvious. Further, we have combined single and 2-DE with MALDI-TOF for analysis of BmHSPs. Ninety kilodalton band excised from 1-DE gel was identified as HSP83 by MALDI-TOF-MS. The immunoblot analysis confirmed the expression of HSP90 in all the instars larvae of B. mori. Heat shock-induced protein spots were excised from 2-DE gels for MALDI-TOF-MS analysis. The Mascot search results are for HSP68, HSC70-1 and HSP70Ba in Pure Mysore, and major HSP70Bbb, HSP68, HSC-3 and HSP83 in NB(4)D(2). Multiple sequence alignment explicit the variations in amino acid sequence between Pure Mysore and NB(4)D(2). Notably, the PMF of spot 2 matched the coding sequence of B. mori and its gene annotation was determined on chromosome 9. With this novel approach, expression of BmHSP90 was confirmed in all the instars and uncovered isoforms of BmHSP70, which provided unequivocal insight to analyze and understand the biological significance in B. mori.