Numerous pre-clinical and clinical studies on reovirus have generated valuable information which supports the use of this orphan virus as an investigational drug for cancer treatment. Reolysin® (pelareorep) is a clinical formulation of the human Reovirus Type 3 Dearing strain. The clinical safety and efficacy of Reolysin® in humans is being tested on an assortment of cancer indications as a mono and/or combination therapy. Reovirus has many inherent characteristics that make it a potential candidate for virotherapy, including: the rapid and natural spread through the haematogenous route, the ability to overcome immunological barriers thereby reaching tumor sites, and being replication-competent. The purpose of this study was to elucidate the bio-distribution pattern of Reolysin® in healthy Sprague-Dawley rats. Following a single 15-min intravenous infusion via the tail vein in Sprague-Dawley rats, the levels of virus genome were determined in 16 organs/tissues by RT-qPCR (Reverse Transcriptase- Quantitative Polymerase Chain Reaction) over a 336 h (Day 15) incubation regime. Consistent with previous studies, maximal reovirus RNA levels were observed in the spleen; indicating its involvement in viral uptake and clearance, followed by heart, ovaries, tail (infusion site), liver and lungs. All the organs/tissues demonstrated unquantifiable levels of reovirus genome at the end of incubation, suggesting substantial to complete viral clearance. Several studies in the last decade have described the use of reovirus for treating ovarian cancers. An increase of reovirus genome in ovaries at 24 h post infection was noted. The results will aid in the design of additional exploratory clinical trials for Reolysin®.