The NSW Environment Protection Authority (EPA), under its statutory responsibility, must identify uncharacterized materials that are suspected to cause environmental harm. Samples are collected during investigations of discoloration of natural watercourses, odors, atmospheric fallout, dumped waste drums with unknown contents, and unexplained deaths or physical distress of plant and animal species. Laboratory investigations are undertaken in order to facilitate the correct handling and disposal of orphan waste, to determine the source of a substance, or to identify the cause of death or distress to flora or fauna. No formal published procedure, similar to toxicity identification evaluation (TIE) protocols for the identification of unknown samples, exists. Such a procedure needs to consider the diverse nature and complexity of the samples and the extremely large number of possible contaminants. A further complication is that the unknown material is often contained in environmental matrices. This article describes a unique procedure developed by the NSW EPA's Environmental Forensic and Analytical Science Section (EFAS) to identify potentially hazardous unknown substances. The procedure is based on a series of hierarchical flow charts that have been converted into an interactive, web-based, expert system. This system addresses the need for a comprehensive, systematic, and documented procedure to provide scientific rigor, acceptability in legal cases, and a tool for training junior chemists. The system has undergone a successful testing and validation program. Examples of its use in environmental forensic investigations conducted by the NSW EPA are provided. Ongoing development is focusing on the use of chemometric techniques to match a substance mixed in an environmental matrix with a pollutant source.