Serum samples collected from 23 flocks of commercial hens from three different companies were tested by enzyme-linked immunosorbent assay (ELISA) for antibodies against infectious laryngotracheitis (ILT) virus, and data were analyzed statistically. Geometric mean titers (GMTs) were compared from hens that were unvaccinated, once-vaccinated, or twice-vaccinated, from single-age farms or multiple-age farms, from molted or unmolted flocks, and from different companies. There were significant differences among the groups compared by vaccination, between the single-age and multiple-age groups, and between the molted and unmolted groups. The GMT of unvaccinated flocks and the GMT of molted flocks that had been vaccinated once as pullets with a chick-tissue-culture-origin (CTCO) live vaccine could not be differentiated. The ELISA is useful to detect and quantitate ILT vaccine-induced antibody, but it does not reliably identify older flocks that were vaccinated at a young age with CTCO vaccine.