Fresh and frozen-thawed rhesus monkey sperm were analyzed for DNA damage using the comet assay and for chromosome damage by cytogenetic analysis after intracytoplasmic sperm injection (ICSI) into mouse oocytes. The percentage of fresh sperm with damaged DNA in ejaculated semen was 0 to 2.7% (n = 5). Conventional cryopreservation and storage in liquid nitrogen caused DNA damage in 25.3% to 43.7% of sperm; when sperm were frozen without cryoprotectants, 52.7% to 92.0% of thawed sperm had DNA damage. However, no significant difference in chromosome damage was found between fresh sperm and frozen-thawed sperm when motile sperm were selected for ICSI. The percentage of sperm with abnormal karyotypes ranged from 0 to 8.3%. The most common structural chromosomal abnormalities in fresh motile sperm and frozen-thawed motile sperm were chromosome breaks or fragments. Our findings suggest that genetically competent frozen-thawed macaque sperm can be selected for fertilization by using only motile sperm for ICSI.