Abstract: Numerical chromosome anomaly was one of the most important kinds of human chromosme diseases by inducing pregnancy loss, miscarriage, infant death, congenital malformations and nerve damage. The present study was to establish a rapid, reliable and reasonable multicolor primed in situ labeling (PRINS) protocol for diagnosing numerical anomaly in human chromosome. First, nuclei of cultured lymphocytes and sperms were labeled with the method of PRINS, and then nuclei of cultured lymphocytes, sperms and other specimen were labeled with the method of updated non-ddNTP-blocking multicolor PRINS technique. The labeling effect of different target sequences and the feature of different fluorochromes were evaluated by experiment. Meanwhile, several parameters of PRINS were optimized to obtain more homogeneous and stable labeling effect. At last, the applicative value of PRINS was evaluated by comparing the clinical effect and labeling characteristics between FISH probe and PRINS. In the present study, several chromosomes were simultaneously marked successfully in the same sperm nucleus within 2.5 hours. And the frequency of one-color-labeling reached 99%. The many advantages, compared with FISH, make PRINS become the first choice in diagnosing diseases related to numerical anomaly in human chromosome.