Leukocyte telomere length is a biomarker of aging-related health risks. Hospitalized preterm infants frequently experience elevated oxidative stress and inflammation, both of which contribute to telomere shortening. Our aim was to examine changes in telomere length during neonatal intensive care unit (NICU) hospitalization in a cohort of preterm infants <32 weeks' gestation. We conducted a longitudinal study of 10 infants (mean gestational age 27 weeks, range 23.5 to 29, at birth). We isolated DNA from dried blood spots and used Real Time Quantitative PCR to measure relative leukocyte telomere length in triplicate at three time points for each participant. From birth to discharge, infants experienced an average decline in relative telomere length of 0.021 units per week (95% CI -0.040, -0.0020; p = 0.03), after adjustment for gestational age at birth. Our results suggest a measurable decline in telomere length during NICU hospitalization. We speculate that telomere length change may convey information about NICU exposures that carry short- and long-term health risks.