Background/Question/Methods Microhabitat preference among primates, which provides them with the niche they need to survive, often conditions primate diversity, abundance, and coexistence. Vegetation alteration and recovery have built heterogeneous forest landscapes that may influence primates’ microhabitat preference. We compared the diversity and size of trees/shrubs and the presence of lianas in 132 sites where we captured the rufous mouse lemur (Microcebus rufus), with that of 240 sites where we did not capture this species, to investigate the aspects of microhabitat structure they prefer. We then examined how this structural preference varies across a heterogeneous landscape of forests with different disturbance levels. Results/Conclusions Overall, microhabitats used by M. rufus differed significantly from unused ones in densities of small size, understory, and midstory plants. Microcebus rufus frequented microhabitats with significantly denser small- and medium-size (DBH 2.5-10 cm) trees/shrubs without lianas in the primary forest and small-size plants (DBH 2.5-4.9 cm) with one liana in other forest types. Compared to the microhabitats they used in the primary forest, the microhabitats in other forest types had lower densities of trees/shrubs with lianas. Additionally, the secondary forests and forest fragments also had significantly lower DBH. Although this variation in microhabitat use may represent an opportunity for M. rufus to live in disturbed habitats, it may expose them to additional threats, affecting their long-term survival. These findings emphasize the need to examine potential changes in microhabitat use among primates living in anthropogenic landscapes, which could help optimize long-term conservation and management of threatened primate species in heterogeneous landscapes.