Wheat (Triticum aestivum L.) is the important and strategic cereal crop for the majority of world’s populations. It is significant staple food of about two billion people. In next few years, world demand for wheat is expected to be 40 percent higher than that of its level today. Keeping in view the importance of the crop research work was conducted in the laboratory of Plant Breeding and Molecular Genetics, University of Poonch Rawalakot, AJK, Pakistan. The aim of the research was to find out the genetic diversity of different wheat lines. In the experiment, 50 different lines of wheat species (Triticum aestivum L.) was used to detect genetic diversity by utilizing Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and biochemical analysis. On the basis of biochemical analysis lines 3111 and 3123 was diverse among 50 lines for antioxidant activity by using DPPH radical and 3135 and 3139 for phenolic contents and for flavonoid 3148 and 3107 was found more promising. Molecular characterization by SDS PAGE showed diversity in three wheat lines 3136, 3138 and 3110. These wheat lines could be our potential lines for future wheat improvement program as they were also promising regarding to the high yields.