The gluten protein composition and expression level influence dough properties and are cultivar and environment dependent. To broaden the knowledge of the durum wheat gluten proteome, three cultivars were compared in two different growing seasons by a proteomic approach. Cultivar-specific and differentially expressed spots in the two years were identified by mass spectrometry. Significant differences were observed among the cultivars: Ofanto showed the lowest protein spot volumes in the high molecular weight (HMW) and low molecular weight (LMW) <35,000 regions and the highest in the LMW 48,000-35,000 region, Latino the lowest in the LMW 48,000-35,000 region, and Simeto an intermediate expression level in both LMW regions. In the warmer year the up-regulation of HMW glutenins, α-gliadins, and a globulin 3 protein and the down-expression of LMW glutenins and γ-gliadins were observed. Among the cultivars, Simeto showed the highest stability across the environments.