A single-strand conformation polymorphism (SSCP) technique was developed for identification of genetic variation among 26 isolates of St. Louis encephalitis (SLE) virus. A 750-bp portion of the envelope gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the products analyzed by SSCP. SSCP reliably identified genetic variation among the isolates from the US, Central and South America. Closely related isolates from a smaller geographic area (Panama) were also distinguishable by SSCP. The sensitivity of this technique was demonstrated by sequencing each of the isolates used; SSCP was capable of discriminating between isolates that had as few as 1-6 nucleotide differences. These results indicate that SSCP has excellent potential as a tool to screen rapidly SLE virus isolates for genetic variation and could be incorporated into molecular epidemiology studies.