<h3>Research Objectives</h3> To examine the relationship between variability of gait spatiotemporal parameters and pain interference in children with hypermobile spectrum disorder/hypermobile Ehlers-Danlos syndrome (HSD/hEDS). <h3>Design</h3> Cross-sectional, prospective study. <h3>Setting</h3> Movement Analysis for Biomedical Innovation and Technology (Mobility) Lab at the University of Wisconsin-Milwaukee, Milwaukee, WI. <h3>Participants</h3> Thirteen children with HSD/hEDS; 3 males, 10 females; 14.2 ± 2.9 years of age, body mass index: 22.0 ± 6.4 kg/m2. <h3>Interventions</h3> Not applicable. <h3>Main Outcome Measures</h3> Coefficient of variation (CV) was used to represent the variability of gait (CV = standard deviation (SD)/mean*100). The CV of step time, step length, gait speed, and cadence from five gait cycles were calculated. CV value greater than 3 SD from the mean was excluded from the analysis. The Brief Pain Inventory interference items related to daily activities (i.e., general activity, mobility, and going to school) were analyzed using a 0-10 scale where 0 is no interference and 10 is complete interference. Associations between gait variability and pain interference scores were analyzed using Pearson's correlation (r). <h3>Results</h3> The participants had CV of step time (n=12, 5.2 ± 9.5 CV%), step length (n=13, 5.5 ± 4.2 CV%), gait speed (n=13, 4.8 ± 2.5 CV%), and cadence (n=12, 5.7 ± 10.2 CV%). Brief Pain Inventory scores comprised general activity (2.5 ± 1.9), mobility (3.4 ± 2.0), and school (2.1 ± 2.8). Greater step length variability was moderately to strongly associated with greater pain interference with general activity (r=0.65, p=0.016), mobility (r=0.61, p=0.026), and going to school (r=0.78, p=0.003). <h3>Conclusions</h3> Increased gait variability was significantly correlated to pain interference during functional activities in children with HSD/hEDS. The variability may be due to altered muscle activation, disrupted proprioceptive feedback, and the instinct to protect from injury and provocative pain. Alterations in gait parameters and increased pain interference may reduce the quality of life. Future studies are needed to examine the mechanisms contributing to gait variability to guide patient-specific interventions for children with HSD/hEDS. <h3>Author(s) Disclosures</h3> The authors have no conflicts of interest to disclose.