This study was carried out to evaluate the effects of washing medium, breed and washing temperature of fresh and frozen-thawed boar sperm on mitochondrial activity and membrane integrity by flow cytometry. More than $80\%$ of fresh sperm washed with mTLP-PVA medium at $20^{circ}C$ exhibited an intact membrane and a functional mitochondrion. With frozen-thawed samples, a large number of sperm showed both damaged membrane $(36.4~46.9\%)$ and nonfunctional mitochondrion $(55.1~71.1\%)$ in the mTLP-PVA and BTS washing media at $20^{circ}C$. There were no breed effects of fresh and frozen-thawed sperm on mitochondrial activity and membrane integrity. The percentages of damaged membrane of fresh and frozen sperm, respectively, were higher at $4^{circ}C$ washing temperature than at $20^{circ}C$ washing temperature in the mTLP-PVA medium. We found that washing medium and washing temperature of fresh and frozen-thawed boar sperm were important for the analyses of mitochondrial activity and membrane integrity by flow cytometry.