Infectious laryngotracheitis (ILT) is a severe acute respiratory disease of chickens caused by ILT virus. To better understand the epidemiology of the disease, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay of the glycoprotein E gene has been developed and utilized to characterize vaccine strains and outbreak-related isolates. Enzymes EaeI and DdeI were used to differentiate the tissue culture origin (TCO) vaccine from chicken embryo origin (CEO) vaccines. Two RFLP patterns were observed with enzyme EaeI, one characteristic of the TCO vaccine and a second characteristic of all CEO vaccines. Three RFLP patterns were observed with enzyme DdeI. Patterns A and B were characterized as single patterns, whereas the type C pattern was a combination of patterns A and B. Analysis of vaccine strains showed the presence of patterns A and C. Pattern A was observed for the TCO vaccine and one CEO vaccine, whereas pattern C was observed for five of the six CEO vaccines analyzed. PCR-RFLP analysis of plaque-purified virus from pattern C CEO vaccine preparations demonstrated the presence of two populations (patterns A and B). Identification of molecularly different populations of viruses within currently used ILT vaccine is the first step to develop better molecular epidemiologic tools to track vaccine isolates in the field.