The aims of this study were to elucidate the detailed anatomy of the abducens nerve in the lateral rectus muscle (LRM) and the intramuscular innervation pattern using Sihler staining. In this cohort study, 32 eyes of 16 cadavers were assessed. Dissection was performed from the LRM origin to its insertion. The following distances were measured: from LRM insertion to the bifurcation point of the abducens nerve, from LRM insertion to the entry site of the superior branch or inferior branch, from the upper border of the LRM to the entry site of the superior branch, from the lower border of LRM to the entry site of inferior branch, and the widths of the main trunk and superior and inferior branches. The single trunk of the abducens nerve divided into two branches 37 mm from insertion of the LRM, and 22 of 32 (68.8%) orbits showed only two superior and inferior branches with no subdivision. The superior branch entered the LRM more anteriorly (P = 0.037) and the superior branch was thinner than the inferior branch (P = 0.040). The most distally located intramuscular nerve ending was observed at 52.9 ± 3.5% of the length of each muscle. Non-overlap between the superior and inferior intramuscular arborization of the nerve was detected in 27 of 32 cases (84.4%). Five cases (15.6%) showed definite overlap of the superior and inferior zones. This study revealed the detailed anatomy of the abducens nerve in the LRM and provides helpful information to understand abducens nerve palsy. Clin. Anat. 30:873-877, 2017. © 2017 Wiley Periodicals, Inc.