Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus (ILTV). To evaluate differential detection of ILTVs belonging to the two types, wild-type or vaccine-type, both causing clinical signs, five PCRs were evaluated to detect wild-type and vaccine-type ILTV in clinical samples. By directly sampling the organs, we aimed to avoid changes in the virus genome and to facilitate a fast diagnosis. The samples were tracheal and spleen homogenates and feather shafts. The latter are easy to collect, nonlethal for the bird, and advantageous for monitoring purposes. We investigated the time interval for vaccine virus detection following commercial vaccination by the vent application, which is successfully practiced in Israel. The study indicated that ILTV amplification from feather shafts was possible in clinical cases for about a one-month period after vaccination. Vaccine strains were identified by nested PCR for the ILTV-gE gene and differed from wild-type ILTV strains by two criteria: (1) While avirulent vaccines could be detected for about a month after the vent application, wild-type virus could be detected, in conjunction with clinical signs, for an unlimited time period; and (2) The ILTV vaccine was present in the bird in minute quantities compared to the wild-type virus. We assessed the virus type that appeared in conjunction with the clinical signs and determined that the clinical signs appeared in conjunction with both molecular forms of ILTV. The vaccine virus-type and the wild-type ILTV differed by their distinct restriction pattern when using the HaeIII restriction enzyme digestion of the nested amplification product.