Aim:Native rabbits in smallholder system are considered as important genetic resources, and the present study was aimed to study the genetic and phenotypic characterization and detection of the maternal origin of the native rabbit populations located at the Middle of Egypt.Materials and Methods:A survey of native rabbit populations was conducted in three governorates (Fayum [FY], Beni Suef [BN], and El Menia [MN]). The phenotypic characterization of rabbits included the profile body of the head, ears, eyes, neck, and legs and the coat colors. The blood samples were collected for genetic characterization based on mitochondrial (cytochrome b) and the microsatellite markers.Results:The phenotypic characterization of the body parts in the three populations was almost similar. The body weight of the mature rabbits in MN Government was significantly heaviest, and the measurements for the main body parts (body length, chest circumference, and abdominal girth) were the highest compared to the two populations. The results of mitochondrial (cytochrome b) analysis revealed that the rabbits from the three governments belonged to lineage A except one animal was recorded as lineage G from MN’s rabbit population. The results of the microsatellite markers revealed that the genetic diversity between the three populations showed genetic interferences; however, a closer genetic relationship was observed between BN and MN than FY. The majority of the genetic diversity was the individual variability.Conclusion:The mitochondrial lineage A is the major lineage in rabbit populations in the area of the Middle Egypt understudy. The genetic populations’ structure is the interferences among the three populations. A large-scale survey should be done on native rabbit populations for the sustainable management and conservation of the local breeds’ genetic resources.