The introduction of the short arm of rye chromosome 1R (1RS) into wheat significantly increased grain yields. Studies have shown that 1RS carries a locus controlling root biomass and improves canopy water status under water-stressed conditions. The general genetic map location of the locus is known but allelic variation would facilitate precise mapping and identification of the responsible gene(s). To this end, six 1RS.1BL translocations from various sources in three wheat backgrounds were tested for root biomass and response to drought but no significant differences among different 1RS arms were observed. The behavior of the standard 1RS.1BL translocation in cv. Pavon 76 in various experiments suggested that wheat chromosome arm 1BS of Pavon 76 may carry a locus for root system plasticity. A set of single substitutions of chromosome 1B from 15 different sources, in the same genetic background of cv. Pavon 76, were tested for root biomass in various experiments. Again, no significant variation among the 1B substitution lines was observed. These results suggest that either no allelic variation at the targeted loci exists, or the sets of lines were biased. While various 1B chromosomes originated from a random sample of wheats, all 1RS arms were imported into wheat from various triticales, perhaps preselecting certain allelic combinations.