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Understanding microhabitat requirements for species vulnerable to anthropogenic threats can provide important information to conservation managers. This may be particularly true for ectotherms, where behaviour and physiology (e.g. digestion, responsiveness and activity patterns) are strongly influenced by thermal conditions of microhabitat retreat sites. Retreat sites selected by south-west carpet pythons (Morelia spilota imbricata) were identified through radiotracking 46 pythons over 3 years. Tree hollows appear to be a very important resource for pythons: 61% (22 of 36 individuals tracked over winter) used tree hollows as retreat sites (56% of all observations in winter), and remained in hollows for an average of 124 ± 49 (range 34 to 210) days. If pythons did not use tree hollows over winter, they found refuge in one of four alternative microhabitats: low vegetation cover (26% of winter observations), ground cover (10%), on tree branches (6%) or in hollow logs on the ground (2%). We tested whether tree hollows provide a thermally distinct environment compared with alternative microhabitats, but found no difference in minimum, average, maximum or range of temperatures recorded between microhabitats. When within tree hollows over winter, pythons had colder daily average and maximum body temperatures (cf. pythons that used other microhabitats), but this did not give them an energy saving (in terms of body condition scores). Pythons ate very little over winter and we predict that animals sequestered within tree hollows do not access prey at this time. Tree hollows provide a critical refuge over winter when python body temperature is low, and their responsiveness is limited, rendering individuals vulnerable to predation by terrestrial predators (e.g. introduced red fox). Destruction of hollows through fire, land clearing, competition with other fauna species and the significant age required for hollows to form in trees all contribute to the decline in availability of this important microhabitat.