A procedure was developed for differentiation of vaccine strains and field isolates of infectious laryngotracheitis virus (ILTV) by restriction fragment length polymorphism (RFLP) of DNA fragments amplified from the genome of ILTV by polymerase chain reaction (PCR). RFLP patterns of viral thymidine kinase (TK) gene, glycoprotein C (gC) gene, glycoprotein X (gX) gene and ICP4 gene amplified from different ILT viruses were compared. The results showed that the vaccine strain of tissue-culture-origin (TCO) could be readily distinguished from other ILT viruses. Moreover, two out of the four field isolates could be differentiated from vaccine strains of chicken embryo origin (CEO); but the remaining two field isolates were identical to the CEO vaccine strains. These results suggested that both vaccine-like and vaccine-unlike ILT viruses were involved in the field outbreak of this disease, and that the PCR/RFLP procedure could serve as a fast and sensitive method for the detection and differentiation of vaccine strains and field isolates of ILT viruses.