PURPOSE: To determine the cutoffs for the interocular difference in retinal nerve fiber layer (RNFL) thickness measured with Cirrus HD-OCT (Carl Zeiss Meditec, Inc) in normal eyes. DESIGN: Observational, clinical study. METHODS: Scans were acquired at 7 academic glaucoma clinics from both eyes of 284 normal subjects using the Optic Disc Cube 200 × 200 protocol. The interocular differences in RNFL thickness were calculated, and normal ranges of interocular differences were determined as the 2.5th and 97.5th percentiles. RESULTS: The average RNFL in the right eye was 0.52 μm thicker than in the left eye; the difference was marginally significant (P = .049). The temporal, nasal, and inferior quadrants had significantly thicker RNFL in the right eye, whereas the left eye showed thicker RNFL in the superior quadrant. The 2.5th and 97.5th percentile interocular difference tolerance limits for average RNFL thickness were -7.9 μm and 8.8 μm, respectively. Although the difference in average RNFL thickness correlated with differences in axial length, disc area, cup-to-disc ratio, and vertical cup-to-disc ratio, only differences in axial length (β = -0.21; P < .001) and disc area (β = 0.17; P < .001) were associated with an interocular difference of average RNFL thickness after adjustment for each other. The interocular difference remained stable despite significant decrease in RNFL thickness with aging. CONCLUSIONS: An interocular difference in average RNFL thickness exceeding 9 μm when measured with the Cirrus HD-OCT in normal eyes may be considered statistically significant asymmetry and may be indicative of early glaucomatous damage.