Genetic diversity, clustering pattern and phylogenetic relationship between Moshtohor rabbits (M-line) as a new synthetic line and their founder line of Spanish V-line and Sinai Gabali were assessed using sixteen microsatellite markers. Moreover, polymorphism based on SNP G/A2464 located in the promoter region of progesterone receptor gene (PGR) was evaluated based on PCR-RFLP method. The French Giant Papillon (FGP) was used as an out-group breed. All the investigated microsatellite loci were polymorphic and the average NO, HO, He, FIS were 6.75, 0.65, 0.80 and 0,083 respectively. M-line rabbits recorded the highest values of NO (5.50), Ho (0.758) and He (0.742). Three genotypes (GG, AA and GA) were detected by genotyping G/A2464 SNP located in the promoter region of progesterone receptor (PGR) gene. M-line recorded the highest values of Ne (1.987), He (0.497) and Polymorphic information content (PIC) (0.373) and showed high potentiality for litter traits improvement by possessing the highest GG genotype frequency (0.120) among the studied populations. A close relationship between M-line and V-line (0.18) was supported by their clustering in one phylogenetic clade and their appearance as one admixed mosaic cluster in population structuring.