This study aimed to investigate the sensory innervation of the anterior eye segment in rats by retrograde tracing with 1,1-dioleyl-3,3,3,3-tetrameth-ylindocarbocyanine, 4-chlorobenesulfonate (FAST Dil) injected into the anterior chamber. In our study, the sensory innervation of distinct elements of the anterior segment of the rat’s eye, i.e. the cornea, ciliary body, iris, and trabecular meshwork, were studied by retrograde tracing using FAST Dil as a tracer. FAST Dil was injected into the anterior chambers of the rat’s eyes. The animals were sacrificed at different time points, i.e., 2, 3, 4, and 5 days after the injection. FAST Dil localization in trigeminal ganglions was studied with a fluorescent microscope. Two days after FAST Dil injection into the anterior chambers, the cornea, the ciliary body, the iris, and the trabecular meshwork were heavily labeled. Neurons in the ipsilateral trigeminal ganglion were also consistently labeled. The number of labeled cells increased over time until 4 days after FAST Dil injection. FAST Dil-labeled neurons could be divided into two parts. Most of the Dil-labeled neurons were concentrated in a sharp, longitudinal, spindle-like stripe, located in the dorso-medial side of the trigeminal ganglion, approximately two thirds of the dorsal portion. The other part of Dil-labeled neurons scattered laterally to the stripe, but just in one third of the dorsal region. Thus, with our preliminary results, we conclude that the primary afferent sensory neurons innervating the rat’s anterior eye segments aggregate in the dorso-medial part of the ipsilateral trigeminal ganglion. It is feasible to identify them using retrograde tracing with FAST Dil anterior chamber injection.