Seed protein extraction is complicated and different seeds need different extraction protocols.  In our quest for a high-resolution peanut seed two-dimensional (2-D) gel proteome map, we utilized and standardized a phenol-based protein extraction protocol for separating proteins on pre-cast 24 cm IPG (pH 4-7) strips and large-format gradient (12-14%) SDS-PAGE.  Employing dry mature seeds of Valencia C (sweet) and Tamspan (bitter), of subspecies fastigiata var. fastigiata, we obtained extremely clean and reproducible protein patterns on 2-D gel, fulfilling the main goal of the present study.  A total of 20 silver nitrate stained protein spots differentially expressed between these cultivars were detected, excised from gel, and analyzed by nano electrospray ionization liquid chromatography mass spectrometry to reveal 18 distinct proteins.  With a good protein extraction and 2-D system in our hands, we also examined the 2-D protein profiles of Georgia Green and NC-7, of subsp. hypogaea var. hypogaea, to distinguish among the four peanut market types.  Allergen Arah3/Arah4 was completely absent in Valencia C unlike Tamspan and NC-7 indicating its superiority in terms of allergen content.  A ca. 30 kDa protein with putative homology to Gly1, a 11s globulin seed storage protein was significantly present in Valencia C and absent in Tamspan, and may be associated to taste.  Galactose-binding lectin proteins with antinutritive properties were absent in Valencia C, Georgia green and NC-7 cultivars and again could be related to their consumer preference.  This study demonstrates the potential application of gel-based proteomics for genotyping (fastigiata and hypogaea botanical types) and differentiating peanut market types having different taste and allergen contents in seeds.