3M(b) Triticum aestivum L. (Mv9kr1) - Aegilops biuncialis Vis. (MvGB642) addition lines were crossed with the Chinese Spring ph1b mutant genotype (CSph1b) to produce 3M(b)-wheat chromosome rearrangements. In the F3 generation, 3M(b)(4B) substitution lines and 3M(b).4BS centric fusions were identified with in situ hybridization using repetitive and genomic DNA probes, and with SSR markers. Grain micronutrient analysis showed that the investigated Ae. biuncialis accession MvGB382 and the parental line MvGB642 are suitable gene sources for improving the grain micronutrient content of wheat, as they have higher K, Zn, Fe, and Mn contents. The results suggested that the Ae. biuncialis chromosome 3M(b) carries genes determining the grain micronutrient content, as the 3M(b).4BS centric fusion had significantly higher Zn and Mn contents compared with the recipient wheat cultivar. As yield-related traits, such as the number of tillers, the length of main spike, and spikelets per main spike, were similar in the 3M(b).4BS centric fusion and the parental wheat genotype, it can be concluded that this line could be used in pre-breeding programs aimed at enriching elite wheat cultivars with essential micronutrients.