Grasslands are among the most imperiled ecosystems in North America. Reasons that grasslands are threatened include conversion to row-crop agriculture, fragmentation, and changes in fire regimes. The reduction of fire processes in remaining prairies has resulted in tree encroachment and establishment in grasslands, further reducing grassland quantity and quality. Grassland birds have been experiencing precipitous population declines in recent decades, commensurate with landscape changes to grasslands. The lesser prairie-chicken (Tympanuchus pallidicinctus Ridgway) is a declining species of prairie grouse of conservation concern. We used second- and third-order habitat selection metrics to test if female lesser prairie-chickens avoid grasslands where trees were present. Our results indicated that female lesser prairie-chickens selected habitats avoiding the nearest trees by 283 m on average, nearly twice as far as would be expected at random. Lesser prairie-chickens were 40 times more likely to use habitats with tree densities of 0 trees ∙ ha− 1 than habitats with 5 trees ∙ ha− 1. Probability of use indicated that lesser prairie-chickens were 19 times more likely to use habitats 1000 m from the nearest tree when compared with using habitats 0 m from the nearest tree. Nest survival was not affected at densities < 2 trees ∙ ha− 1; however, we could not test if nest survival was affected at greater tree densities as no nests were detected at densities > 2 trees ∙ ha− 1. Avoidance of trees could be due to perceived increased predation risk, reduced habitat quality, or a combination of these potentially confounding factors. Preventing further establishment and expansion of trees in landscapes occupied by lesser prairie-chickens could contribute to the continued persistence of the species. Additionally, restoring grasslands through tree removal may facilitate conservation efforts for grassland species such as the lesser prairie-chicken by improving habitat quality and promoting expansion of occupied range.