Assessing durum wheat genomic diversity is crucial in a changing environmental particularly in the Mediterranean region where it is largely used to produce pasta. Durum wheat varieties cultivated in Portugal and previously assessed regarding thermotolerance ability were screened for the variability of coding sequences associated with technological traits and repetitive sequences. As expected, reduced variability was observed regarding low molecular weight glutenin subunits (LMW-GS) but a specific LMW-GS allelic form associated with improved pasta-making characteristics was absent in one variety. Contrastingly, molecular markers targeting repetitive elements like microsatellites and retrotransposons – Inter Simple Sequence Repeat (ISSR) and Inter Retrotransposons Amplified Polymorphism (IRAP) – disclosed significant inter and intra-varietal diversity. This high level of polymorphism was revealed by the 20 distinct ISSR/IRAP concatenated profiles observed among the 23 individuals analysed. Interestingly, median joining networks and PCoA analysis grouped individuals of the same variety and clustered varieties accordingly with geographical origin. Globally, this work demonstrates that durum wheat breeding strategies induced selection pressure for some relevant coding sequences while maintaining high levels of genomic variability in non-coding regions enriched in repetitive sequences.