Background. The lack of segmentation algorithms operative across optical coherence tomography (OCT) platforms hinders utility of retinal layer measures in MS trials. Objective. To determine cross-sectional and longitudinal agreement of retinal layer thicknesses derived from an open-source, fully-automated, segmentation algorithm, applied to two spectral-domain OCT devices. Methods. Cirrus HD-OCT and Spectralis OCT macular scans from 68 MS patients and 22 healthy controls were segmented. A longitudinal cohort comprising 51 subjects (mean follow-up: 1.4 ± 0.9 years) was also examined. Bland-Altman analyses and interscanner agreement indices were utilized to assess agreement between scanners. Results. Low mean differences (-2.16 to 0.26 μm) and narrow limits of agreement (LOA) were noted for ganglion cell and inner and outer nuclear layer thicknesses cross-sectionally. Longitudinally we found low mean differences (-0.195 to 0.21 μm) for changes in all layers, with wider LOA. Comparisons of rate of change in layer thicknesses over time revealed consistent results between the platforms. Conclusions. Retinal thickness measures for the majority of the retinal layers agree well cross-sectionally and longitudinally between the two scanners at the cohort level, with greater variability at the individual level. This open-source segmentation algorithm enables combining data from different OCT platforms, broadening utilization of OCT as an outcome measure in MS trials.