Infectious laryngotracheitis (ILT) is a highly contagious disease of chickens and is responsible for significant economic losses in the poultry industry worldwide; it is caused by Gallid herpesvirus-1 (GaHV-1), commonly known as infectious laryngotracheitis virus (ILTV). Experimental evaluation of ILTV strains is fundamental to identify changes in virulence that can contribute to the severity and spread of outbreaks and consequently influence the efficacy of vaccination. Several criteria had been utilized to determine the degree of virulence associated with ILTV strains. The objectives of this study were to compare the levels of virulence of the standard United States Department of Agriculture (USDA) challenge strain with a contemporary outbreak-related strain (63140) and to evaluate the efficacy of individual criteria to identify changes in virulence. Broilers were inoculated with increasing infectious doses of each strain. The criteria utilized to evaluate virulence were clinical signs of the disease, mortality, microscopic tracheal lesions, trachea genome viral loads, and antibody titers. Clinical signs scores were a useful parameter to define the peak of clinical disease but did not reveal differences in virulence between strains. Similarly, trachea microscopic lesion scores or levels of serum antibody titers were parameters that did not reveal obvious differences in virulence between strains. However, mortalities and increased viral genome loads in trachea of chickens inoculated with lower (log ₁₀ 1 to 2) infectious doses clearly differentiated 63140 as a more-virulent ILTV strain. This study provides the framework to compare the virulence level of emerging ILTV isolates to the now-characterized USDA and 63140 strains.