Background <p>Molecular sex determination is a valuable tool for an effective population management in captive bred endangered species. The molecular approaches are particularly valuable in species with limited sexual dimorphism. Moreover, the utilization of accessible non-invasive samples from newborn animals enables early sex determination beneficial for the animal population management and planning.</p> Methods and results <p>A rapid molecular sex determination method using PCR with specific primers targeting the male-specific <i>SRY</i> locus and the control <i>c-myc</i> gene present in both sexes was developed. The method was validated using DNA samples from blood of adult males and females of Sumatran orangutan (<i>Pongo abelii</i>), western lowland gorilla (<i>Gorilla gorilla gorilla</i>), and silvery gibbon (<i>Hylobates moloch</i>). Then we applied the method to non-invasive samples from four newborns: two Sumatran orangutans, one western lowland gorilla, and one silvery gibbon. DNA of the newborns was isolated from umbilical cord material obtained <i>post partum</i> and/or hair. Additionally to the above mentioned species, the method was also successfully validated in adult males and females of chimpanzee (<i>Pan troglodytes</i>), pig-tailed macaque (<i>Macaca nemestrina</i>), Campbell’s monkey (<i>Cercopithecus campbelli</i>), mantled guereza (<i>Colobus guereza</i>), Geoffroy’s spider monkey (<i>Ateles geoffroyi</i>), and black-and-white ruffed lemur (<i>Varecia variegata</i>).</p> Conclusions <p>We demonstrated that the umbilical cord material can be used for molecular sex determination in newborns and that the <i>c-myc</i> gene can serve as a control gene in molecular sex determination, providing an alternative to other commonly used methods.</p>