This study was conducted to evaluate 32 spring wheat genotypes in a field experiment as well as at the molecular level using EST-SSR and SSR markers, to estimate genetic diversity. Results of field evaluation revealed a significant variation between the tested genotypes. The graphic analysis based on Euclidean distances, using the estimated values for each of the five traits separately, divided the 32 genotypes into two main groups, and each group was divided into small sub-clusters. The 32 wheat genotypes were divided into nine primary groups by a cluster analysis based on molecular data, with similarity coefficients ranging from 0.320 to 0.890, with an average of 0.687. The PIC values of the EST-SSR primers ranged from 0.111 to 0.665. But the PIC values of the SSR primers ranged from 0.17 to 0.737. Based on the results obtained here, we have identified six new advanced breeding lines that can be recommended for use in future wheat breeding programs, four of them L7,L8,L31 and L38to improve grain yield productivity as well as , L20 to develop dwarf or semi-dwarf cultivars , and L 3 for breeding to short-season cultivars.