Background: Little is known about the stability of the gut microbiota in multiple sclerosis (MS) over time. Objectives: To determine if the gut microbiota is stable over time in a cohort of individuals with pediatric-onset MS and monophasic acquired demyelinating syndromes (ADS). Methods: The study cohort consisted of individuals from 4 Canadian and 1 American site enrolled in the Canadian Pediatric Demyelinating Disease Network study who provided ?2 stool samples, reported diet using the Block Kids Screener and had body mass index measured. DNA was extracted from stool samples, and the V4 hypervariable region of the 16S rRNA gene was amplified, sequenced on the Illumina MiSeq platform, and clustered into amplicon sequence variants (ASVs). Microbiota composition was assessed using alpha (Shannon, inverse Simpson) and beta diversity (unweighted Unifrac) metrics and ASVs (genus-level) were analysed using nonparametric microbial interdependence test and linear mixed effect models with sex and diagnosis as covariates. Results: We included 36 individuals, including 18 with MS (72% female) who had a mean age at symptom onset of 14.4 (SD 3.9) years, and 18 with ADS (44% female) who had a mean age of onset of 7.6 (SD 4.0) years. Fifteen of the MS and 16 of the ADS participants provided 2 stool samples (averaging 9.5 and 12.5 months apart, respectively), while the remainder provided a third sample at a later timepoint. Mean ages at first stool sample procurement were 18.0 (SD 5.0) years for the MS participants and 13.5 (SD 5.3) years for the ADS participants. Two MS and all the ADS participants were disease-modifying drug (DMD) naïve, and 4 MS participants stopped or switched DMD between samples. Body mass index and dietary metrics did not change meaningfully across stool samples. Alpha and beta diversity did not differ between stool samples (Friedman, Wilcoxon signed-rank, PERMANOVA all p>0.2). Of the 9 genus-level ASVs assessed (importance values ?0.03), 1 genus within the Gemellaceae family varied significantly over time (linear mixed models p=0.001). Changes in DMD did not alter interpretation of findings. Conclusions: The gut microbiota composition in individuals with pediatric-onset MS and related demyelinating syndromes exhibited stability over time, when at least two stool samples were procured over a 9 to 12-month period. Further work includes additional exploration of cohort characteristics. Findings also deserve replication in a larger cohort.