The European ground squirrel (Spermophilus citellus), an endangered small mammal endemic to Central and Southeastern Europe, has an unfavourable-inadequate conservation status in Romania. This study assessed the species density of active burrow openings within six areas of the Natura 2000 site ROSAC0108 Lunca Mureșului Inferior in Western Romania. Distance sampling line transect counts of active burrow openings were conducted for two consecutive seasons. Survey design resulted in interannual difference in effective strip width, encounter rate and detection probability. Higher densities were obtained using systematic parallel transects with complete spatial coverage of known colonies than using random line transects in potentially suitable habitat. This study provides insights into methods for estimating population density by using distance sampling approach to counting active burrow openings.