Tetraclinis articulata (Vahl) Masters (Cupressaceae) is a woody tree species indigenous to North Africa. It is also found occasionally in Malta and Spain showing significant adaptability to different bioclimatic conditions. Due to its economic and ecologic values, this tree is affected by human and environmental disturbance leading to habitat fragmentation and genetic diversity loss. In this study, we aimed to promote the conservation of T. articulata , by measuring the allelic richness of a representative sample of 129 germplasms. Results showed differences in allele distribution. Inter–simple sequence repeat (ISSR) markers and the advanced maximization strategy implemented in PowerCore software were deployed to establish the first core collection of T. articulata . A core collection containing 10.07% out of the 129  T. articulata  germplasms was established. The representativeness of the core collection was evaluated and validated by different methods. The genetic diversity parameters or molecular traits (the observed number of alleles (Na), the effective number of alleles (Ne), Shannon’s information index ( I ), and Nei’s expected heterozygosity (He) showed similarities between the core collection and the entire collection averaging at Na = 1.712, Ne = 1.398, I  = 0.395, and He = 0.253 and Na = 1.970 Ne = 1.363, I  = 0.382, and He = 0.238, respectively. The analysis of molecular variance indicates that most of the genetic variation in Articulata core collections resided within populations (96%), with lesser amounts among populations (4%). Two clusters were identified with STRUCTURE and validated with unweighted pair group method with arithmetic mean dendrogram. The identified collection will be useful for conservation and breeding programs.