Genetic resources of crop plants are essential for crop breeding. They are conserved in gene banks in form of a large numbers of accessions. These accessions harbor allelic variants of agronomically important genes and molecular tools allow a rapid assessment of this allelic diversity. Here, we have screened a collection of 1005 wheat gene bank accessions for powdery mildew resistance and a molecular characterization for functional alleles at the wheat powdery mildew resistance locus Pm3 was carried out mostly on the resistant accessions. The two analyzed sets of accessions consisted of 733 accessions originating from 20 different countries and 272 landraces originating specifically from Afghanistan. The Pm3 haplotype (indicating the presence of a Pm3-type of gene, susceptible or resistant) was found to be abundantly present in both sets. The accessions with a Pm3 haplotype were further screened for the presence of the functional Pm3a to Pm3g alleles using allele-specific molecular markers. Pm3b and Pm3c were the most frequently found alleles while the other five alleles were detected only in few accessions (Pm3d, Pm3e, Pm3f) or not detected at all (Pm3a, Pm3g). The data further showed that Pm3b is the major source of Pm3-mediated powdery mildew resistance in wheat accessions from Afghanistan. Susceptible allelic variants of Pm3 were found to be widespread in the wheat gene pool. The presented molecular analysis of Pm3 alleles in a diverse set of wheat accessions indicates that several alleles have defined geographical origins. Possibly, the widespread Pm3b and Pm3c alleles evolved relatively early in wheat cultivation, allowing their subsequent diffusion into a broad set of wheat lines.