Premature infants constitute a risk group for thiamin deficiency but only little is known about their thiamin status. The aim of the present study was to investigate the thiamin status of premature infants by determination of thiamin diphosphate (TDP) and to identify risk factors for low TDP concentrations. In a prospective, longitudinal study TDP was determined by HPLC in whole blood in the first days of life and approximately every 2 weeks. Demographical data, weight gain, type of nutrition and thiamin intake were recorded. A total of 111 premature infants were included at the Children’s Hospital of the University of Cologne, Germany from May 2009 until December 2010 and 222 blood samples were analysed. TDP concentrations showed an age-dependent decline (age 0–10 d, mean TDP = 110.6 ng/ml; age 11–20 d, mean TDP = 95.4 ng/ml; age 21–103 d, mean TDP = 33.6 ng/ml). There was no significant difference between males and females. Young gestational age and low birth weight were associated with low TDP concentrations. No infant was diagnosed with thiamin deficiency. The current nutritional regimen in our hospital did not lead to thiamin deficiency in the study cohort. Further research is required to evaluate how TDP concentrations are regulated in premature infants.