The human interest in exotic animal breeds in the agriculture sector led to the deterioration of local breeds. Microsatellite markers are important tools to determine the genetic status of breeds and populations. This study aimed to detect the genetic situation among the Egyptian native rabbit breed (ENR) along the Nile River in rural areas. The survey covered 804 km (50 geographical points) to collect 410 biological samples from three populations: native Delta Egypt rabbits (NDER, n = 153), native Middle Egypt rabbits (NMER, n = 124), and native Upper Egypt rabbits (NUER, n = 133). Across 27 markers, a total of 284 alleles were observed in this study, and about 63% of them were highly polymorphism-formative with no significance for Hardy-Weinberg equilibrium (HWE). The highest values of the mean number of alleles (MNa), private alleles (Pa), and allelic richness (Ar) were recorded in the NUER population (13.519, 21, and 6.762, respectively). The observed heterozygosity (Ho)< expected (He) in two populations (NDER and NMER). In contrast, the NUER population (Ho >He). Values of genetic variability showed that population genetic variability increased in the south direction (NMER and NUER). In the north (NDER), the current study recorded a bottleneck with a high significant value of inbreeding coefficient (FIS) 0.418 vs. 0.145 for NUER in the south. This study found that the ENR situation is critical for increasing the north. While it characterized stability in the south direction.