The red-tailed phascogale (Phascogale calura) is an endangered dasyurid from southwestern Western Australia that has been bred in captivity since 2001. It is an annual breeder that has a restricted breeding period, and males only participate in a single breeding season. We examined the reproductive biology of female red-tailed phascogales, assessing methods for detection of estrus, variation in the timing of breeding in captivity across facilities and years, and reproductive success. Body mass and pouch changes were useful indicators of estrus, whereas assays for fecal estradiol and progesterone did not allow for accurate prediction of ovulation. Females mated with multiple males and matings occurred over a period of at least 5 days, with females storing sperm in the lumen and crypts of the oviduct. Births tended to occur in July at Alice Spring Desert Park, with births at Adelaide Zoo occurring from early June to late August. The predictable births at Alice Springs are similar to those observed with Antechinus, but with flexibility remaining in the reproductive strategy of the species as observed at Adelaide. Of the 146 breeding females at Alice Springs, 127 females produced 846 pouch young, of which 68% were weaned. A female bias was observed in weaned young. Information gained from this study has been incorporated into the captive-breeding program for this species.