Cadmium (Cd) is a non-essential heavy metal having toxic effects on all living organisms. Durum wheat (Triticum durum Desf.) is widely used in human diets but has the potential to accumulate Cd. It also has a high level of genetic diversity, which may be exploited to develop cultivars with low Cd content. We aimed to perform marker-assisted selection and validate previously identified Cd markers in durum wheat germplasm for use in the investigation of accessions that accumulate low grain Cd content. We assessed 130 durum wheat accessions phenotypically and using three different molecular markers. Grain Cd contents of the studied germplasm varied 4.91-fold (26.2–128.7 μg/kg) with an average of 58.2 μg/kg. Landraces showed lower average values of grain Cd content than cultivars. Three molecular markers (usw47, Cad-5B and KASP marker Cad-5B) were used to differentiate high and low Cd accumulating lines. Results showed high correlation and successfully classified the accessions to the expected high or low Cd level; 87 accessions showed the low Cd alleles, and 43 accessions the high Cd alleles, except for five accessions with the usw47 marker that showed heterozygous status. A significant correlation coefficient (r = 0.944*) was observed among the three molecular markers. Based on molecular markers, 96.2% of the accessions were classified accurately. The KASP assay was highly effective in successfully separating low from high Cd content accessions and could be used as a molecular tool in durum wheat breeding programs, with less cost and time, targeting reduced grain Cd levels. The results of this study will allow durum wheat breeders to accelerate their progress to select suitable genotypes with the desired alleles.