A collection of 220 durum wheat breeding germplasms, originating from ICARDA and CIMMYT breeding programs, was assessed to explore phenotypic and genotypic diversity and to identify informative molecular markers for several agro-physiological traits under drought conditions for two cropping seasons (2017–2018, 2018–2019). A large phenotypic diversity based on agro-physiological traits was detected. Based on genotype-by-trait (GT) biplot analysis, several trends in germplasm adaptation to drought conditions were identified. The majority of ICARDA germplasms that were most favored by grain yield, 1000-kernel weight (TKW), normal difference vegetative index (NDVI), plant height (PH), and peduncle length (PL) were clearly separated from most CIMMYT germplasms that most characterized late in heading (DH) and maturity (DM) and higher canopy temperature (CT). These findings indicate that the breeding lines originally developed from ICARDA may possess tolerance traits to drought conditions that are absent in those developed in CIMMYT. The start codon targeted (SCoT) was applied for genetic relationship analysis of 220 breeding lines. The percent of polymorphism ranged from 50% (SCoT25) to 100% (SCoT2) with an average of 72.22%. The SCoT-based cluster analysis identified five different groups corresponding to the genomic constitution in durum germplasm, which was in part confirmed by principal coordinate analysis. Grouping of germplasms using multivariate clustering methods allowed identification of breeding lines with outstanding characteristics under rainfed conditions. Some clear trends in adaptation of germplasms to rainfed conditions based on phenotypic data were observed, which were mostly dependent on geographical difference of germplasms.