Abstract Objectives To establish reference intervals for thyroid stimulating hormone (TSH) and free thyroxine (FT4) at 3–6weeks of age in very low birth weight (VLBW) infants with the Beckman Coulter Unicel DxI 800 by gender, birth weight (BW) and gestational age (GA) subgroups. Design and methods A 4year retrospective cohort of 308 VLBW infants (GA=27.9weeks, BW=992.3g) was studied. All blood samples for TSH and FT4 were analyzed using the modified fTSH2 (TSH) and two-step competitive enzyme immunoassay (FT4). Reference intervals were evaluated according to the most recent Clinical and Laboratory Standards Institute (CLSI) guidelines. Results The study provides non-parametric 95% reference intervals with associated 90% confidence intervals for FT4 and TSH derived from 308 infants screened at a median of 31days. The reference intervals for this population are TSH=1.14–11.04mIU/L and FT4=10.9–21.4pmol/L. TSH statistically differed according to birth weight (<1000g vs 1000–1499g) while FT4 differed according to gender and gestational age at time of testing (<32weeks vs ≥32weeks); however, these differences were not clinically significant and a single reference interval for each analyte is reported. Conclusion The results of this study highlight the importance and complexity of establishing appropriate reference intervals for thyroid function testing for the preterm population.