Frozen semen quality is one of many factors that promote successfully of artificial insemination. Preservation at 5ºC is one of the steps of semen freezing to obtain high fertility sperm after added with the extender. This study was aimed to determined the sperm quality (motility, viability, and membrane integrity), MDA level and DNA damage of post-thawed sperm after being preserved at 5ºC for different duration. Bali cattle bull semen collected by artificial vagina. Macroscopic and microscopic evaluation of ejaculate was conducted first. Qualified semen was diluted in Tris Aminomethan-Egg yolk and then devided into two treatment group: preserved at 5ºC for 4 hours (first group) and 22 hours (second group), and continued to processed into frozen semen, and stored overnight. Pre-freezing and post-thawed of semen analysis was conducted based on SNI: 4869-1:2017 of The National Standardization Agency of Indonesia. Measurement of levels of Malondialdehyde (MDA) was conducted by spectrophotometry at 532 nm wavelength, meanwhile measurement of sperms DNA damage percentage was performed by Toluidine blue staining. The result of showed that quality of post-thawed sperm (motility, viability, and membrane integrity) was higher (P<0.05), and MDA level and DNA damage were lower (P<0.05) in preservation at 5°C for 22 hours compared to those of 4 hours. It could be concluded that preservation at 5°C longer (22 hours) means sperm had longer chance to adapt with the extender, this implies the higher quality and lower lipid peroxidation.