This work focused on investigating the performance of thirteen newly wheat breeding lines compared with two check varieties under two salinity conditions; 6000 and 9000 ppm. The objectives were to determine salt-tolerant and sensitive genotypes using diverse tolerance indices. Additionally, to assess the genetic variability, heritability, genetic gain and genetic diversity among breeding lines using ISSR markers, protein electrophoresis and superoxide dismutase isozymes (SOD). The phenotypic and genotypic coefficients of variation displayed maximum values for number of spikes/m2, grain yield and number of grains/spike. Seven tolerance indices were calculated; SSI, TOL, MP, GMP, STI, YI and YSI for all evaluated genotypes. The highest scores of all indices were registered by G10 followed by G3, G13, G7 and G1; on the contrary, the lowest values were recorded by G12, G8 and G5. Cluster analysis was performed based on tolerance indices and the genotypes were divided into four groups ranged from highly salt-tolerant to highly sensitive genotypes. ISSR-PCR analysis displayed 105 DNA fragments using 6 selected primers detected 77 polymorphic bands and 28 monomorphic bands. Protein electrophoresis revealed that the number of bands for all genotypes ranged from 11 to 14 with molecular weights ranged between 6 to 130 kDa under salinity stress conditions. Unique bands of molecular weights 17 and 20 kDa were detected just for G3 and G4. SOD patterns indicated more intensive bands for G3 and G4 under salinity stress.