An association of retroviruses and especially HIV has been reported in SALS, but CNS tissues have not been directly tested. To be valid, direct testing should be performed in light of the highly variable distribution of neuropathology from one patient to another and the degradable nature of RNA viruses. We aimed to seek direct evidence of retrovirus and especially HIV in SALS by real-time RT-PCR. We tested 20 SALS spinal cords in our repository specifically created for RNA preservation. We extracted RNA from anterior horns and surrounding tissue scrape from each sample and validated RNA quality. We indexed our search to neuropathologic degeneration and tested multiple areas in many of the nervous systems. We used real-time RT-PCR to a highly conserved region in the highly conserved pol gene and established the lowest limit of detection using a positive control. HIV-1 pol gene sequence was not detected in any of the 20 SALS nervous systems. Our assay was able to detect down to 60 viral copy numbers. In conclusion, we could not find direct evidence of retroviral/HIV activity in SALS CNS to support a relationship. However, our method could miss novel retroviruses with non-homology in the pol gene.