Powdery mildew is a severe disease in wheat. In barley, durable resistance exists, based on non-functionality of the Mlo gene. As a model to analyse the effects of mutagenesis in the homoeologous Mlo genes of wheat, we developed mlo-based powdery mildew resistance in tetraploid durum wheat. To obtain Mlo mutations, we screened a TILLING population developed in tetraploid wheat "Kronos" for which the captured exome sequence of > 1500 lines is available. This resulted in 23 mutants for Mlo-A1 and 26 non-redundant mutants for Mlo-B1. Two Mlo-A1 and four Mlo-B1 mutants were crossed to obtain eight F2 mutant lines that showed a range of phenotypes from susceptibility to full resistance. Pot experiments under semi-field conditions confirmed the resistance levels for six of the mutants without any signs of adverse pleiotropic effects. Resistance ranking was similar across six powdery mildew isolates, indicating no isolate specificity of the mlo-based resistance. The effect of mutations in the Mlo-B1 gene was stronger than in the Mlo-A1 gene, probably reflecting differences in wild-type Mlo gene expression levels. Strong partial resistance effects were observed with single mlo-B1 mutations hence, revealing a dosage effect of mlo mutant alleles. Two of the four mlo-B1 mutations (W163* and P335L) were very strong; however, the highest combined effect was observed with the MloA-P335S/MloB-P335L combination, suggesting that non-functional, but full-length Mlo proteins might have the strongest effect compared with nonsense mutations. Our results show that mlo-based resistance might offer possibilities to introduce durable protection in tetraploid wheat against powdery mildew.