Two-dimensional gel electrophoresis has been extensively used for separation and characterization of proteins. A protocol was established for two-dimensional gel electrophoresis (2-DE) seed proteome map of six sorghum genotypes including non-pigmented lines IS 3477, IS 33095, IS 7005 and pigmented lines are 2898, IS 7155 and IS 1202 in the pH range of 3-10. Proteins extracted from flour in a TCA/Acetone buffer were focused after cup-loading onto IPG strips. Successful separation in the second dimension was achieved using gradient gels in a horizontal SDS-PAGE system. The 2-D gels of six genotypes stained with Coomassie Brilliant Blue (CBB) which were resolved a total of 116, 140, 109, 98, 80 and 102 protein spots in IS 3477, IS 33095, IS 7005, IS 2898, IS 7155 and IS 1202 respectively. Furthermore, we have identified different protein spots by using PD-Quest software among the genotypes which were included Up-regulation protein spots (162), Downregulation protein spots (483), Qualitative protein spots (91), Quantitative protein spots (109), Common protein spots (542) and Specific protein spots (93). The presented 2- D patterns and identifications will be used to describe proteome differences between cultivars.