Abstract Background Iron deficiency (ID) in childhood is linked to adverse neurodevelopmental outcomes in the long-term. Extremely premature infants (EPI; born < 28 weeks gestation) are more susceptible to ID due to a multitude of factors including lower iron reserves at birth, rapid growth, immature erythropoiesis, and multiple phlebotomy tests during hospital stay. Therefore, professional societies advise prophylactic iron therapy for these infants from 2-6 weeks age until 6-12 months. However, there is paucity of data on the iron status in EPI receiving this prophylactic iron supplementation. Objectives To investigate the prevalence and risk factors associated with ID in EPI. Design/Methods A retrospective cohort study analyzed a Provincial database of EPIs born between 2005-2018. Infants with congenital malformations, chromosomal anomalies and blood disorders were excluded. All included infants received prophylactic iron supplementation from 2-4 weeks of chronological age that was recommended to continue at discharge until 9-12 months corrected age (CA). At 4-6 months CA, these infants underwent blood testing. ID was defined as serum ferritin < 20mcg/L at 4 months CA or SF < 12mcg/L at 6 months CA. Through a univariate analysis using single-variable logistic regression, factors linked to ID were identified. Those with a p-value < 0.20 progressed to a multivariable model, retaining variables with a p-value < 0.05. Results Of 146 infants, 45.9% had ID. ID group had lower mean ferritin (16.4 µg/L vs 50.0 µg/L, p < .001) and reticulocyte hemoglobin equivalent (28.4pg vs 31.6pg, p < .001) compared to the non-ID group. ID prevalence reduced from 59.7% in 2005-2011 to 40.3% in 2012-2018 cohort. Exclusive or partial breastfeeding at 4-6 months CA was protective against ID (Odds: 0.2, p = 0.003). Iron therapy at 4-6 months CA was not protective in the final model. Notably, 27.4% stopped iron therapy before 4-6 months CA, and exclusive formula-fed infants had notably lower iron intake than breastfed infants (66.1% vs 99.1%, p = 0.006). Conclusion Nearly half of the EPI exhibited ID at 4-6 months CA despite prophylactic iron supplementation. About a quarter discontinued therapy before reaching this age. Breastfeeding at this age was protective against ID. This high prevalence of ID in EPI underscores the need of future studies to discern strategies to mitigate ID in this vulnerable population.