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Abstract The mitogenomes of 29 Egyptian river buffaloes were sequenced, characterized, and investigated for nucleotide variations in the mitochondrial protein-coding genes as well as the possible effects of variations on the translated proteins. The mitogenome was 16,357–16,359 base pairs in length and contained the 37 genes found in a typical mammalian mitogenome. Its overall base composition was A: 33.1%, C: 26.6%, G: 13.9%, and T: 26.4%. The start and stop codons variety as well as the 22 tRNA anticodons that have been detected here are similar to that of genus Bubalus members. Sequence alignment indicated the presence of 44 nucleotide variations along the protein-coding genes. 35 of the variations were synonymous while the others (9) were non-synonymous. Investigations in the non- synonymous variations indicated that only 2 variations in MT-ATP8 (m.7874A>T) and MT-ND5 (m.12083C>T) have led to deleterious amino acid substitutions in ATP8 (N36Y) and ND5 (P112L), respectively. Results will help subsequent studies to research mitochondrial polymorphisms that can be related to important utility traits for the genetic improvement of river buffalo herds. Also, results of mitogenome based phylogenetic analysis indicated that river buffalo and swamp buffalo may belong to two distinct species |