The pygmy slow loris Nycticebus pygmaeus is a little-studied primate endemic to Vietnam, Laos, southern China and eastern Cambodia. Our study aimed to gain local knowledge on the distribution and ecology of, and threats to, the species by interviewing hunters, traders and wildlife protection staff, and to verify this information using a spotlighting survey in three major reserves in Mondulkiri Province, Cambodia. Encounter rates of pygmy loris were assessed along 29 transects (129.5 km), yielding observations of 26 individuals. Mean encounter rates were 0.40 km-1 in Seima Protection Forest, 0.10 km-1 in Phnom Prich Wildlife Sanctuary and 0.00 km-1 in Mondulkiri Protected Forest. Informants had knowledge of where populations occurred, their diet, sociality and habitat preferences. Widespread large population declines were reported and informants linked this to high hunting pressure, particularly in 2001 and 2002. In late 2008 and 2009 we resurveyed three transects that had high encounter rates in early 2008 and failed to detect any lorises. Local informants reported high hunting pressure during the previous wet season in two of these sites, and a gold mine development was underway in the third site. Urgent actions are required to address these population declines and to assess the conservation status of pygmy lorises throughout eastern Cambodia.