Sewage impacted soil, sludge and water samples were studied to understand the occurrence and formation of thiosteranes and to determine the relevance of these compounds as tracers for sewage input into the environment. Soils were collected from wastewater irrigation fields (Wroclaw, Poland), water from the Nexapa River Basin (Mexico), which also received wastewater and wastewater treatment plant(WWTP) effluent, and water and sludge from the Norman WWTP (USA) at different treatment stages. Thiosteranes represented a high proportion of the steroid fraction in the Wroclaw irrigation field and the Nexapa River Basin samples. Small amounts of thiosteranes were found in anaerobically digested sludge from the Norman WWTP. A good correlation between coprostanone and thiosterane concentrations suggests thiosteranes were produced by stanone sulphurization under anoxic conditions. Thiosterane stability under anoxic and suboxic conditions indicates their potential use as tracers for environmental input of sewage products or land application of sewage sludge.