Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes severe respiratory disease in chickens. Vaccines for the control of ILTV can be produced in embryonated chicken eggs, however the tropism, distribution and pathogenesis of ILTV in ovo is incompletely understood. Ultramicroscopy techniques such as light-sheet fluorescence microscopy (LSFM) allows for high resolution 3D imaging of large tissue sections up to 5 mm in depth. This study examined sites of ILTV infection in ovo using LSFM and compared these results to established detection techniques such as qPCR and histopathology. Replicate embryonated eggs were inoculated at day 14 of incubation with 1000, 3000 or 10000 plaque forming units (PFU) of an mCherry labelled ILTV mutant via either the amniotic or allantoic cavity. Eggs were incubated for 3 days before examination of the embryo and embryonic annexes for LSFM, qPCR and histopathology. Detection of ILTV infection by qPCR was variable between eggs but was consistently high in the amniotic and allantoic membranes. The inoculation route had limited impact on the location of infection, and the inoculating dose did not impact the viral load within the annexes. The embryo mostly remained refractory to infection, where detection of ILTV was sporadic. Evidence of embryonic infection was also difficult to detect by histopathology. Visualisation of anatomical structures was successful with LSFM and infection was detected within the trachea of one embryo from an egg inoculated via the amniotic cavity with 1000 PFU. Taken together, these data indicate that ILTV infection concentrates to the embryonic annexes in ovo and is difficult to detect in the embryo by any method. Nevertheless, LSFM is a useful technique for providing a 3D structural context for in ovo examination alongside traditional detection methods.