Sexes of cranes cannot be distinguished visually due to the morphological similarities between male and female. To facilitate the protection program for the endangered birds, red-crowned cranes in particular, genomic DNA was extracted from feather samples and a PCR-based sexing method was established using three primer combinations of avian sex-specific EE0.6 sequence. The specificity of the PCR was confirmed by amplifying genomic DNA from 22 known-sex cranes of seven species. After 30 cycles of amplification, easily distinguishable single bands for males and two bands for females were displayed by agarose gel electrophoresis. The sexes of 50 unknown-sex cranes of seven species were successfully identified using the PCR. The noninvasive PCR-based sexing method can not only facilitate the human-assisted breeding of cranes, but also provide useful reference for accurate sexing of other endangered birds.