Genetic diversity was assessed among 32 advanced wheat breeding lines included in the National Uniform Wheat Yield Trials (2006-07) of Pakistan using molecular (DNA) and biochemical (SDS-PAGE) markers. Of the 72 RAPD primers used for initial screening, 15 were found polymorphic. A total of 140 bands (61.4% polymorphic) were generated by the 15 random decamer primers. Genetic similarity coefficients ranged from 0.81 to 0.94 for rainfed and from 0.70 to 0.93 for the normal seeding date group. Cluster analysis using the unweighted pair group method of arithmetic averages (UPGMA) clustered the 32 advanced wheat breeding lines into one major and three small groups. Maximum level of polymorphism (90%) was observed for the primer OPA-05. Lines N9 and N11 showed the least genetic similarities (0.70-0.82 and 0.71-0.83, respectively) with rest of the lines studied. Line RF1 had the maximum similarity (0.81-0.94) with other lines. Wheat lines included in the normal seeding date were relatively distantly related than those in the rainfed group. Seed storage protein analysis produced 19 subunits ranging from 29-120KDa. Similarity coefficients ranged from 0.53 to 1.0 for the normal seeding date and from 0.47 to 1.0 for the rainfed group. High molecular weight subunits (particularly 120KDa) showed greater polymorphism than the lower molecular weight subunits. Narrow genetic base was observed in wheat lines included in the rainfed group. DNA fingerprinting of advanced breeding lines may help to avoid release of varieties with narrow genetic base in the future.