In Mediterranean environments, water-deficit stress that occurs before anthesis significantly limits durum wheat (Triticum turgidum L. ssp. durum) production. Stress tolerant and stress sensitive durum varieties exhibit genotypic differences in their response to pre-anthesis water-deficit stress as reflected by yield performance, but our knowledge of the mechanisms underlying tolerance is limited. We have previously identified stress responsive durum microRNAs (miRNAs) that could contribute to water-deficit stress tolerance by mediating post-transcriptional silencing of genes that lead to stress adaptation (e.g. miR160 and its targets ARF8 (auxin response factor 8) and ARF18). However, the temporal regulation pattern of miR160-ARFs after induction of pre-anthesis water-deficit stress in sensitive and tolerant varieties remains unknown. Here, the physiological responses of four durum genotypes are described by chlorophyll content, leaf relative water content, and stomatal conductance at seven time-points during water-deficit stress from booting to anthesis. qPCR examination of miR160, ARF8 and ARF18 at these time-points revealed a complex stress responsive regulatory pattern, in the flag leaf and the head, subject to genotype. Harvest components and morphological traits measured at maturity confirmed the stress tolerance level of these four varieties for agronomic performance, and their potential association with the physiological responses. In general, the distinct regulatory pattern of miR160-ARFs among stress tolerant and sensitive durum varieties suggests that miRNA-mediated molecular pathways may contribute to the genotypic differences in the physiological traits, ultimately affecting yield components (e.g. the maintenance of harvest index and grain number).