AbstractOur study aimed to incorporate 60 newly acquired spring durum wheat genotypes into the Prague Gene Bank collection, focusing on 54 experimental breeding research genotypes and their technological quality contributions for conservation and future use. We analyzed a total of 11 field and 6 technological parameters of grain quality. Additionally, we identified the composition of high molecular weight glutenin subunits (HMW-GSs) in 13 accessions with contrasting technological characteristics. Two different extraction procedures were employed to detect HMW-GSs: one based on total protein extraction and the other on gliadin pre-extraction. The tested parameters exhibited a significant range of variability, with the relative standard deviation ranging from 2.1% for starch content to 96.7% for the gluten index. Additionally, six breeding research genotypes exhibited high technological grain quality comparable to the two modern durum wheat cultivars. Two accessions: M90-99–2 and IG 142076 showed above-average Zeleny sedimentation values, probably positively influenced by the presence of HMW-GSs A1: (2*). Genotypes PI 675012 and IG 142039 displayed the highest grain stability in technological parameters. Both extraction methods also detected the specific subunit B1: (6 + 8*) in the genotype IG 142039. Preserving these genotypes thus represents a substantial expansion of genetic resources in the genebank.