Thiamine pyrophosphate (TPP) is the major biologically active form of thiamine (vitamin B1). This cross-sectional study assessed whole-blood thiamine pyrophosphate concentration (WBTPPC) in boarding school students in the Southern Region of Papua New Guinea. Sample size for each of the five boarding schools was calculated using the 'proportionate to population size' cluster sampling technique. The 'Clin-Rep' reagent kit was used for the extraction of thiamine pyrophosphate from whole blood. Reverse phase high performance liquid chromatography with post-column derivatization was used to determine the thiamine pyrophosphate concentration. Informed consent was obtained from 468 students, mean age 17.7 +/- 1.5 years. The gender distribution of these students was 274 (58.5%) males and 194 (41.5%) females. The median and interquartile range of WBTPPC for all students was 95.41 microg/l (82.27-113.55). Severe to marginal status of thiamine deficiency was present in 6.4% of all the students. The mean WBTPPC for female students was significantly lower than that for the male students (p < 0.001), with a mean difference of 14.17 microg/l (95% CI of the difference: 9.85-18.50). Severe to marginal status of thiamine deficiency was present in 9.8% of female students and 4.0% of male students. The data strongly support the need for effective implementation and monitoring of food fortification legislation in Papua New Guinea. Withdrawal of fortification or suboptimal thiamine fortification of rice and other cereal products in Papua New Guinea would have serious negative public health implications, especially among students in boarding schools.