We conducted the first comprehensive breeding season survey of Bengal Florican within the Tonle Sap region (19,500 km2). During 2005/06 and 2006/07 we systematically sampled 1-km squares for territorial males. Bengal Florican were detected within 90 1-km squares at a mean density of 0.34 males km−2 which, accounting for unequal survey effort across grassland blocks, provides a mean estimate of 0.2 males km−2. Based on 2005 habitat extent, the estimated Tonle Sap population is 416 adult males (333–502 ± 95% CI), more than half of them in Kompong Thom province. Tonle Sap grasslands are rapidly being lost due to intensification of rice cultivation and, based on satellite images, we document declines of 28% grassland cover within 10 grassland blocks between January 2005 and March 2007. Based on mean 2005 population densities the remaining grassland may support as few as 294 adult male florican, a decline of 30% since 2005. In response to these habitat declines almost 350 km2 of grassland have been designated as protected areas, set aside for biodiversity and local livelihoods.