Introduction: Domestic sheep (Ovis aries), one of the earliest domesticated animal species, have played a major role in human nutrition and clothing and have expanded to nearly all regions of the world through human migrations. The genetic diversity of domestic sheep is crucial for understanding their domestication history and evolutionary relationships. This study was conducted to investigate the genetic diversity and phylogenetic structure, with particular emphasis on indigenous Iranian breeds.
 
Materials and Methods: In the present study, a total of 933 mitochondrial genome nucleotide sequences of domestic sheep, representing 202 breeds from 49 countries, were retrieved from the NCBI Genome Database. The dataset was classified into 14 geographical regions. The sequences were aligned using MAFFT v7.526 on the Galaxy v25.0.2 platform with the FFT-NS-2 algorithm. Nucleotide composition, genetic distance, and the number of transition and transversion mutations were calculated using MEGA12 software. Genetic diversity parameters, including nucleotide diversity (π), mean number of nucleotide differences (k), number of haplotypes, polymorphic sites, and total number of mutations, were estimated using DnaSP 6. A phylogenetic tree for the entire dataset was constructed in MEGA12 using the Neighbor-Joining algorithm, the resulting tree was visualized using iTOL v7.Molecular variance analysis was performed using Arlequin 3.5.Neutrality tests to assess nucleotide diversity deviations from the neutral evolution hypothesis and to detect signatures of natural selection (Tajima’s D and Fu’s Fs) were conducted in DnaSP 6. The obtained haplotypes were matched against the reference genome in the National Center for Biotechnology Information (NCBI) using BLAST. Haplotype networks for Iranian breeds were constructed using PopART1.7, while NETWORK 4.1was used for the entire dataset.
 
Results and Discussion: The results revealed significant haplotype diversity across different regions and Iranian breeds, highlighting patterns of genetic differentiation within and between populations. The mean nucleotide composition indicated an AT-rich sequence. The level of conserved sequences across all samples was 0.861. A total of 686 haplotypes were identified globally, including 57 haplotypes in Iran. The haplotype network, with over 500 unique haplotypes and the predominance of haplogroup B in Europe and Africa, haplogroup A in Asia, confirmed the Fertile Crescent origin and global dispersal. Haplogroup B, with a frequency of 528, was the most prevalent across geographical regions, while haplogroup D, with only 3 occurrences, was the least frequent. The highest haplogroup diversity was observed in geographical regions 2, 3, and 5 (West Asia, Central Asia and East Asia). Haplogroup E was detected in Georgia, China, Afghanistan, and Palestine, while haplogroup D was observed in Turkey and Georgia. Haplogroups A and B were present in all geographical regions. In Iran, the frequencies of haplogroups were 52% for A, 30% for B, and 18% for C. Out of 72 samples, 57 haplotypes were identified, of which 80% were unique. Mitochondrial genome sequences from Iranian local breeds exhibited haplogroups A, B, and C. Haplogroups A and B were observed in Moghani and Maku breeds, A and C in Baluchi and Shirazi Gray breeds, A in Shal breed, and A, B, and C in Afshari and Qaregol breeds. Tajima’s D and Fu’s Fs statistics were negative in 13 geographical regions, indicating population expansion, particularly in regions with ancient domestication histories. The phylogenetic tree revealed evolutionary relationships between Iranian breeds and global populations. Most studied breeds clustered in closely related branches, suggesting gene flow between different geographical regions and historical admixture. Asian populations exhibited higher haplotype diversity compared to European populations. Molecular variance analysis revealed that the majority of genetic variance (71.53%) occurred within populations, indicating high genetic diversity at the local level. However, 23.68% of the variation was observed among four continental groups, reflecting significant genetic differentiation across continents (Asia, Europe, Africa, and South America). The high statistical significance of these indices confirms the presence of a robust genetic structure and significant differentiation between continents and populations, likely reflecting the influence of geographical, historical, or ecological factors on genetic dispersal.
 
Conclusion: This study demonstrated that the mitochondrial genetic diversity of domestic sheep has been shaped by the intertwined processes of domestication, human migrations, and subsequent gene flow. A significant genetic structure was observed among geographical regions, with high local diversity primarily attributed to within-population differences. Haplogroup patterns, particularly the predominance of haplogroup B and the geographical distribution of haplogroups A and C, indicate rapid expansion following domestication and the influence of ancient trade routes. These findings underscore the importance of conserving high genetic diversity, particularly in regions like Iran with unexplored genetic potential, and highlight the need for more advanced genomic studies. To preserve the genetic diversity of domestic sheep and sustainably utilize their genetic potential, conservation strategies focused on local populations with high diversity, especially in regions like Iran, should be designed and implemented.