Barley powdery mildew (BPM) is caused by the pathogen Blumeria hordei (Bh) and can lead to severe yield loss. Plant pathologists are looking for new sources of resistance to BPM. Barley accessions, including the wild subspecies Hordeum vulgare subsp. spontaneum (Hvs), are stored in many gene banks and are often a valuable source of economically important characteristics. The wild barley Hvs could be a valuable resistance source for BPM. The aim of the presented investigation was to detect new sources of BPM resistance in 81 accessions of Hvs collected in Jordan (46), Lebanon (24), and Libya (11). European differential isolates of BPM were used, and resistant single plant lines were selected for use from fifteen accessions from Jordan and Libya. These resistant single plant lines were tested for the presence of specific resistance genes using a differential set of Bh isolates. Hypotheses about the presence of specific resistance genes were made by comparing the reaction spectra of the tested lines with those of differential lines. After an analysis of the obtained results, it was concluded that all 31 tested single plant lines of Hvs had genes for resistance that are not represented in the barley differential set for resistance genes to Bh. Twenty-six lines of Hvs selected from accessions originated in Jordan and Libya showed resistance reactions to all isolates used. These lines will be further tested as new sources of effective resistance and used in barley prebreeding programs.