Background: Alterations in sperm mitochondrial DNA (mtDNA) affect the functions of some OXPHOS proteins which will affect sperm motility and may be associated with asthenozoospermia. The purpose of this study was to investigate the correlation between 7599-bp and 7345-bp < /em> sperm mtDNA deletions and asthenozoospermia in Jordan. Methods: Semen specimens from 200 men including 121 infertile and 79 healthy individuals were collected at the Royal Jordanian Medical Services In-vitro fertilization (IVF) units. The mtDNA was extracted followed by mtDNA amplification. Polymerase chain reaction (PCR) was conducted for the target sequences, then DNA sequencing was performed for the PCR products. Chi-square, Fisher's and Spearman's tests were used to calculate the correlation. Results: The results showed a significant correlation between the presence of 7599-bp < /em> mtDNA deletion and infertility where the frequency of the 7599-bp < /em> deletion was 63.6 % in the infertile group compared to the fertile 34.2% (p<0.001, (OR=3.37, 95% CI=1.860 to 6.108)). Additionally, the sperm motility showed a significant association with the frequency of the 7599-bp < /em> deletion (p=0.001, r=-0.887). The 7345-bp < /em> mtDNA deletion showed no assoctiation with the infertility (p=0.65, (OR=0.837, 95% CI=0.464-1.51)) or asthenozoospermia (p=0.98, r=0.008). Conclusion: We demonstrated a significant correlation between asthenozoospermia and the 7599-bp < /em> mtDNA deletion but not the 7345-bp mtDNA deletion in the infertile men in Jordan. Screening for deletions in sperm mtDNA can be used as a pre-diagnostic molecular marker for male infertility.