Gibbons and siamangs (Hylobatidae) are well-known fortheir rapid chromosomal evolution, which has resultedin high speciation rate within the family. On the otherhand, distinct karyotypes do not prevent speciation,allowing interbreeding between individuals in captivity,and the unwanted hybrids are ethically problematic as allgibbon species are endangered or critically endangered.Thus, accurate species identification is crucial for captivebreeding, particularly in China where studbooks areunavailable. Identification based on external morphologyis difficult, especially for hybrids, because speciesare usually similar in appearance. In this study, weemployed G-banding karyotyping and fluorescence insitu hybridization (FISH) as well as a PCR-basedapproach to examine karyotypic characteristics andidentify crested gibbons of the genus Nomascus fromzoos and nature reserves in China. We characterizedand identified five karyotypes from 21 individuals ofNomascus. Using karyotypes and mitochondrial andnuclear genes, we identified three purebred speciesand three hybrids, including one F2 hybrid between N.gabriellae and N. siki. Our results also supported thatN. leucogenys and N. siki shared the same inversion onchromosome 7, which resolves arguments from previousstudies. Our results demonstrated that both karyotypingand DNA-based approaches were suitable for identifyingpurebred species, though neither was ideal for hybrididentification. The advantages and disadvantages ofboth approaches are discussed. Our results furtherhighlight the importance of animal ethics and welfare,which are critical for endangered species in captivity.