In this study we performed a-systematic sequence analysis of 7 mitochondrial genes (cytochrome oxidase I, cytochrome oxidase II, cytochrome oxidase III, adenosine triphosphate synthase6, ATP synthase8, cytochrome b and tRNA(His)) in 64 infertile men suffering from asthenospermia (n = 31) in comparison to normospermic infertile men (n = 33) from Tunisian population. A total of 92 nucleotide substitutions in sperm mitochondrial DNA were found; 88 of them were previously identified and reported in the human mitochondrial DNA database (www.mitomap.org) and 4 were novel. We also detected in 4 asthenospermic patients a double novels mutations, the first was found in COXII gene (m.8021 G/A) that was absent in normospermic infertile men. This mutation substituting the Isoleucine at position 146 to Valine in a conserved amino acid in the transmembrane functional domain of the protein. And the second was detected in the tRNA(His) gene (m.12187C>A) this mutation was found in homoplasmic state and was absent in normospermic patients. It was conserved throughout evolution and affects a wobble adenine in the T-loop region at the 54 codon of mitochondrial tRNA(His). (C) 2014 Elsevier Inc. All rights reserved.