Gibbons and siamangs (Hylobatidae) are well-known for their rapid chromosomal evolution, which has resulted in high speciation rate within the family. On the other hand, distinct karyotypes do not prevent speciation, allowing interbreeding between individuals in captivity, and the unwanted hybrids are ethically problematic as all gibbon species are endangered or critically endangered. Thus, accurate species identification is crucial for captive breeding, particularly in China where studbooks are unavailable. Identification based on external morphology is difficult, especially for hybrids, because species are usually similar in appearance. In this study, we employed G-banding karyotyping and fluorescence in situ
hybridization (FISH) as well as a PCR-based approach to examine karyotypic characteristics and identify crested gibbons of the genus Nomascus
from zoos and nature reserves in China. We characterized and identified five karyotypes from 21 individuals of Nomascus
. Using karyotypes and mitochondrial and nuclear genes, we identified three purebred species and three hybrids, including one F2 hybrid between N. gabriellae
and N. siki
. Our results also supported that N. leucogenys
and N. siki
shared the same inversion on chromosome 7, which resolves arguments from previous studies. Our results demonstrated that both karyotyping and DNA-based approaches were suitable for identifying purebred species, though neither was ideal for hybrid identification. The advantages and disadvantages of both approaches are discussed. Our results further highlight the importance of animal ethics and welfare, which are critical for endangered species in captivity.