Because of continental-scale declines of grassland birds over the past century, conservation agendas are focused on increasing understanding of grassland bird ecology and habitat associations. Shortgrass prairie is a unique grassland ecosystem maintained, in part, by Black-tailed Prairie Dogs (Cynomys ludovicianus). The Mountain Plover (Charadrius montanus) and western Burrowing Owl (Athene cunicularia hypugaea) are species of conservation concern known to be associated with prairie dog colonies. We estimated abundance of Mountain Plovers and Burrowing Owls in three habitats within the Colorado shortgrass prairie ecosystem—prairie dog colonies, grassland not occupied by prairie dogs, and dryland agriculture. Further, we investigated habitat associations of Mountain Plovers and Burrowing Owls at multiple landscape scales. We estimated 8,577 Mountain Plovers (95% CI: 7,511–35,130) and 3,554 Burrowing Owls (95% CI: 3,298–8,445) in eastern Colorado. Mountain Plover density on prairie dog colony plots ( = 2.26 birds per 100 ha, 95% CI: 2.15–5.13) was significantly higher than densities on either grassland ( = 0.23,95% CI: 0.17–1.76) or dryland-agriculture plots ( = 0.45,95% CI: 0.44–0.53). Burrowing Owl density on prairie dog colony plots ( = 3.04 birds per 100 ha, 95% CI: 2.82–6.92) was significantly higher than densities on either grassland ( = 0.044, 95% CI: 0.041–0.12) or dryland-agriculture plots (no Burrowing Owls detected). Our results suggest that increased prairie dog colonies would positively influence the abundance of Mountain Plover and Burrowing Owl.