Artificial insemination is the one of the most used biotechnologies in animal production. Although well established and used worldwide, some critical steps such as thawing still need improvement. Therefore, we aimed to investigate the effect of thawing temperature on cryopreserved sperm and to evaluate sperm viability after storage for 8 hours. Frozen semen from Holstein (Hol, n=5) and Nelore (Nel, n=5) bulls were thawed at 37°C or at 4°C. After thawing, the samples were kept in the same temperature up to 8 hours. Samples were evaluated at 0, 2, 4, 6 and 8h for total (TM) and progressive motility (PM) by CASA (Hamilton Thorne Biosciences, Beverly, Massachussetts – USA) and membrane (MI) and Acrosome Integrity (AI) by flow cytometry (AMNIS Flow Sight, Amnis Corp., Setattle, WA). Data were analyzed by ANOVA using a 2X2 factorial experimental design, based on breed (Hol or Nel) and thawing temperature (4°C or 37°C), and their interactions. Data were compared among groups at the same time point and within group as repeated measure. TM was affected by breed and time (P0.05) when Nel (0h= 62.2% vs 8h =65.5%) and Hol (0h= 43.8% vs. 39.6%) semen was thawed at 4°C. Regarding PM, except for Hol semen thawed at 37°C that had a decrease (P0.05) and were preserved up to 2h. After 4 h there was a decrease in AI and MI on Hol and Nel semen thawed at 37oC (P0.05). No interactions were found between breed and treatment (P>0.005) for any of the assessments. In conclusion, Hol and Nel semen are susceptible to be thawed and stored at 4oC or 37oC until 8h post thawing. However, 4°C thawing/storage, is able to maintain the sperm quality for longer storage time than 37oC. Further studies in vitro and in vivo are needed to confirm such fertilization potential.