Unlocking resistance genes in genbank collections are of prime importance for securing sustainable crop production. In this regard, the Serbian GenBank barley collection, comprising 93 local landraces and 36 commercial cultivars and elite barley breeding lines, was screened for novel resistances to powdery mildew (Blumeria graminis f. sp. hordei) using a set of 28 isolates with a wide spectrum of virulences/avirulences. No line was resistant to all the isolates, but one and three accessions showed resistance to 27 and 26 isolates, respectively. Twenty landraces (21.51 %) and ten cultivars (27.78 %) exhibited resistance to 50 % of the isolates. Infection type 2 was most frequent among resistant accessions. Nine B. graminis isolates were sufficient for gene postulation in 73 barley lines. In total, thirty-five different resistance spectra were recorded and the following known resistance genes were postulated namely, Mlra, Mlh, Mla12, Mla7(Mlu), Mlg, MlLa, Mla6, Mla7, Mlt, Mla22, Mlat, Mla1, Mlk. The majority of resistance profiles was constituted by only one line. Unidentified genes alone or in combination were proposed for twenty landraces and six cultivars. This report demonstrated that the barley collection held at the Serbian GenBank could be exploited as a new source for powdery mildew resistance.