Developing a standardized survey methodology to census and estimate the size of wintering populations is a main conservation priority for the endangered Little Bustard. We present a standard and repeatable methodology to census wintering Little Bustard populations at a regional scale, and the first statistically reliable population estimate of a Little Bustard wintering population in Spain. We carried out two surveys, in 2003 and 2004, using UTM 10 km × 10 km squares as census units, which were subjected to stratified sampling over the species' potential distribution range in the region of Madrid (Central Spain). Only the areas of potentially suitable habitat within squares of known winter presence of the species were considered. The species' winter distribution in Madrid was fairly constant between years, showing a fragmented pattern in three main nuclei. The number of Little Bustards observed was consistent between surveys (752 and 786 birds, respectively), with birds grouped in an average of 32 flocks (1 to 350 birds, mean = 27.9 birds). No between-winter differences in flock size were observed, although differences between sectors were significant. The highest densities were observed in the Tagus valley (mean density = 2.9 birds km−2, 293 birds), followed by north-eastern farmland (mean density = 0.6 bird km−2, 269 birds). The estimated size of the Little Bustard population wintering in the Madrid region was 1,051 (95% CI = 1,043–1,231). This calculation was based on recorded population density, measured as the number of birds per square kilometre, and considers the potential habitats available in each sector. We propose this methodology as adequate for surveying any Little Bustard wintering area, at both regional and smaller spatial scales. Survey results are consistent with previous tentative estimates for the region of Madrid, and with available information on Little Bustard movements. Sixty-five per cent of squares with Little Bustard presence were outside any protected area, which makes the wintering Little Bustard population of Madrid highly vulnerable to habitat disappearance and may affect the viability of breeding nuclei whose individuals winter in the region.