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Handheld spectral-domain optical coherence tomography (SD-OCT) can provide insights into the complex interactions occurring at the vitreoretinal interface in retinopathy of prematurity (ROP) to enhance our understanding of ROP pathology.To characterize vitreous bands in premature infants with use of handheld SD-OCT.Prospective cohort study conducted from July 7, 2015, to February 28, 2017, at 2 university-based neonatal intensive care units. Seventy-three premature infants who required routine ROP screening examination were recruited. Informed consent was obtained from all legal guardians. Trained graders who were masked to the clinical assessment analyzed each SD-OCT scan of the right eye for vitreoretinal findings. A third trained grader mediated disagreements.Associations between the presence of vitreous bands in premature infants with ROP diagnoses and the presence of other vitreoretinal SD-OCT findings were investigated.Of the 73 infants recruited, 6 infants’ parents withdrew their children from the study, and 2 infants were too hemodynamically unstable for imaging, leaving a total of 65 participants. Of these, 32 (49%) were female, 36 (55%) were white, 10 (15%) were Hispanic, 3 (5%) were Native American, 4 (6%) were African American, 4 (7%) were Asian/Pacific Islander, and 8 (12%) were other. The mean (SD) gestational age was 28 (2.7) weeks, the mean (SD) birth weight was 997 g (286 g), and the mean (SD) postmenstrual age at imaging was 34 (3) weeks (mean [SD] total of 3  imaging sessions). Comparing the 24 infants (37%) who had a right eye vitreous band at any time with the 41 (63%) who did not, no difference in mean birth weight, gestational age, postmenstrual age at imaging, sex, or race/ethnicity was identified. No associations with ROP stage (eg, in 6 [25%] infants with vitreous bands vs 4 [9.8%] in those without; P = .23), presence of plus disease (2 [8%] vs 2 [5%]; P = .84), or type 1 ROP (3 [12%] vs 3 [7%]; P = .66) were identified. Vitreous bands were associated with epiretinal membrane detected on SD-OCT (P = .001) with an odds ratio of 9.4 (95% CI, 2.8-31.3) in 15 [62%] infants with vitreous bands vs 6 [15%] in those without. Vitreous bands were also associated with cystoid macular edema (in 15 [62%] infants with vitreous bands vs 1 [27%] in those without; P = .005) with an odds ratio of 4.5 (95% CI, 1.5-13.3).In this study, the development of vitreous bands was associated with both cystoid macular edema and epiretinal membrane. These findings suggest a tractional pathogenesis to these entities among premature infants. This study did not find a direct association between vitreous bands and severe ROP. Additional study is needed to determine whether vitreous bands represent subclinical hyaloidal organization leading to retinal detachment in advanced ROP.