A nested reverse transcription-polymerase chain reaction (RT-PCR) was employed for detection of bovine viral diarrhea virus (BVDV) in 59 semen samples collected from farms of known history of BVDV infection. The results indicated that 6 (10.2%) and 8 (13.6%) net and extended semen samples, respectively, were culture positive and 53 (89.8%) and 51 (86.4%) were culture-negative. On the other hand, 10 (16.9%) and 10 (16.9%) net and extended semen samples, respectively, were nested RT-PCR-positive and 49 (83.1%) and 49 (83.1%) were nested RT- PCR-negative. Ten semen samples were collected from BVDV-free animals to be used as negative control samples and they tested negative by culture, nested RT-PCR and double PCR. The re- vised sensitivity and specificity of nested RT-PCR in net and extended semen samples were 100.00 %. On the other hand, the sensitivity of culture technique in net and extended semen samples were 60.00% and 80 %, respectively, while the specificity were 100% in both net and extended semen, The results indicated that nested RT-PCR is reliable, rapid, highly sensitive, and specific for accurate detection of BVDV in semen samples. According to the best of our knowledge, our work is the first to report a nested RT-PCR assay that has proved to be efficient in detecting the presence of BVDV in semen. Finally, we recommend the use of nested RT-PCR assay as a supplemental diagnostic tool for detection and identification of BVDV in semen.