To determine whether retinal nerve fiber layer (RNFL) atrophy in the afferent visual pathway may complement existing tools used to describe and characterize differences across MS subtypes.Optical coherence tomography-measured RNFL values were compared over two years in 35 patients (70 eyes) with optic neuritis (ON) as a clinically isolated syndrome (CIS); 39 patients (78 eyes) with relapsing remitting MS (RRMS); and 7 patients (14 eyes) with secondary progressive MS (SPMS).RNFL comparisons involving eyes without ON yielded greater differences between MS subtypes than ON-affected eyes. Overall RNFL values in non-affected eyes were reduced in SPMS patients (83.4 μm), relative to CIS (101.2 μm) (p=0.0009), and RRMS patients (103.7 μm) (p=0.001); and temporal RNFL atrophy was greater in RRMS (64.4 μm) eyes as compared to CIS eyes (73.2 μm) (p=0.02). In ON-affected eyes, RNFL atrophy was greater in SPMS patients (39.5 μm) than CIS patients (58.1 μm) (p=0.03), and in RRMS patients (48.2 μm) relative to CIS patients (p=0.05). RNFL values did not change significantly for any MS subtype during the two-year duration of the study.RNFL thickness may represent a structural marker, which can help distinguish MS subtypes, because the extent of atrophy is commensurate with disease progression. RNFL comparisons between non-affected eyes revealed greater differences between CIS, RRMS, and SPMS patients relative to ON-affected eyes, because the impact of prior ON may supplant the effects of disease subtype.