This study evaluates and compares habitat preferences and spatial niche breadth and overlap between two sympatrically occurring species, the sand cat (Felis margarita) and Rüppell's fox (Vulpes rueppellii), in a desert landscape of central Iran. A field survey from 2014 to 2016 was conducted to collect occurrence points for the two species as well as to identify their structural characteristics of used habitats in the occurrences points. Jacobs' index as a measure of habitat preference, Shannon and Levins' indices as measures of niche breadth, MacArthur, Levins and Pianka's indices as measures of niche overlap were calculated and interpreted. The results of this study showed that the sand cats are specifically restricted to sand dune and sabulous habitat type, whereas Rüppell's foxes have wider ecological amplitude. Sand cats only prefers sand dunes while Rüppell's foxes were found to prefer foothills, badlands, and sand dunes. Rüppell's foxes therefore had a wider niche breadth compared to the sand cats. The asymmetric MacArthur and Levins indices yielded a higher value of niche overlap for the sand cats compared to Rüppell's foxes, while the symmetric Pianka's measure of niche overlap was relatively high for both species. Such habitat preference and niche segregation between the two species may be a result of their feeding habits or the physical protective structure of their habitats attributes.