Behind each eye of the chicken resides a unique lymph tissue, the Harderian gland, for which RNA sequencing (RNA-seq) analysis is novel. We characterized the response of this tissue to Newcastle disease virus (NDV) in two inbred lines with different susceptibility to NDV across three time points. Three-week-old relatively resistant (Fayoumi) and relatively susceptible (Leghorn) birds were inoculated with a high-titered (107EID50) La Sota strain of NDV via an oculonasal route. At 2, 6, and 10 days post infection (dpi) Harderian glands were collected and analyzed via RNA-seq. The Fayoumi had significantly more detectable viral transcripts in the Harderian gland at 2 dpi than the Leghorn, but cleared the virus by 6 dpi. At all three time points, few genes were declared differentially expressed (DE) between the challenged and nonchallenged birds, except for the Leghorns at 6 dpi, and these DE genes were predicted to activate an adaptive immune response. Relative to the Leghorn, the Fayoumi was predicted to activate more immune pathways in both challenged and nonchallenged birds suggesting a more elevated immune system in the Fayoumis under homeostatic conditions. Overall, this study helped characterize the function of this important tissue and its response to NDV.