Identifying male and female echidnas is challenging due to the lack of external genitalia or any other differing morphological features. This limits studies of wild populations and is a major problem for echidna captive management and breeding. Non-invasive genetic approaches to determine sex minimise the need for handling animals and are used extensively in other mammals. However, currently available approaches cannot be applied to monotremes because their sex chromosomes share no homology with sex chromosomes in other mammals. In this study we used recently identified X and Y chromosome-specific sequences to establish a non-invasive polymerase chain reaction-based technique to determine the sex of echidnas. Genomic DNA was extracted from echidna hair follicles followed by amplification of two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametolog (CRSPY) and anti-Müllerian hormone Y-gametolog (AMHY)) and the X chromosome gene (anti-Müllerian hormone X-gametolog (AMHX)). Using this technique, we identified the sex of 10 juvenile echidnas born at Perth Zoo, revealing that eight of the 10 echidnas were female. Future use of the genetic sexing technique in echidnas will inform captive management, continue breeding success and can be used to investigate sex ratios and population dynamics in wild populations.