The current study aimed to detect the genetic variability of mitochondrial DNA (mtDNA) cytochrome B (CYTB) gene in 80 native Egyptian rabbits (NER) belonging to three populations in three different agriculture regions (Delta, Middle, and Upper Egypt) and also investigate the origin of these breeds by phylogenetic relationship analysis. A total of 62 haplotypes were recorded among the three NER populations. The native Upper Egypt rabbits (NUER) expressed the highest number of haplotypes, mutations, polymorphic sites, and haplotype diversity (23, 55, 51, and 1.00, respectively). While it gave the lowest values of nucleotide diversity (0.0262) and Tajima's D (−0.01435), the percentage of mutual haplotypes was 4.8 % between the Middle and Delta Egypt populations. The phylogenetic analysis showed that there were more separated haplotypes of NUER compared to other populations. The same finding was also observed when supported by 31 sequences of different rabbit breeds retrieved from the GeneBank database. A mutual haplotype was observed between native Delta Egyptian rabbits (NDER) and New Zealand (NWZ) rabbits. The results of this study shed light on the importance of indigenous breeds in rural areas through mtDNA, which contributes to finding sustainable strategies to conserve and improve genetic resources in Egyptian rural areas