To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T8 plant, having uidA (or gus) driven by the barley endosperm-specific B1-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T4 plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F1 progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F2 seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F2 plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F3 and F4 generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.