Female Tasmanian devils (TDs) Sarcophilus harrisii and spotted-tailed quolls (STQs) Dasyurus maculatus were monitored to assess changes in plasma progesterone and faecal oestrogens/progestagens, vaginal smears and qualitative changes in pouch appearance during the oestrous cycle. Pouch condition was characterized based on size, colour and secretions, and was found to accurately reflect reproductive status, being significantly correlated with changes in both sex steroids and vaginal cytology. During the follicular phase, pouch redness and secretions were maximal, and associated with increased sex steroid concentrations, a karyopyknotic index of >90% and the onset of copulation. Post-ovulation, pouches became wet and deep and developed a glandular appearance; plasma progesterone/faecal progestagen concentrations remained high and sustained throughout the luteal phase. These features were identical during the pregnant and non-pregnant oestrous cycle. This study demonstrated that pouch appearance is a reliable physical indicator of the stage of oestrous in the TD and STQ, and provides an alternative non-invasive method for evaluating the ovarian cycle of these threatened species. This technique can be readily applied to monitor individuals in free-ranging or captive populations, and will aid as a practical tool for improved breeding management.