Serum 25-hydroxyvitamin D /25OHD/ levels in humans are determined primarily by environmental factors such as UV-B radiation and diet, including vitamin D intake. Although some genetic determinants of 25OHD levels have been shown, the magnitude of this association has not yet been clarified. The present study evaluates the genetic contribution to total- /t-25OHD/ and free-25OHD /f-25OHD/ in a representative sample of the Hungarian population (n = 462). The study was performed at the end of winter to minimize the effect of sunlight, which is a major determinant of serum vitamin D levels. Single nucleotide polymorphisms (SNPs) of five genes playing major roles in vitamin D metabolism were investigated (NADSYN1, DHCR7, GC, CYP2R1 and CYP24A1). The selected SNPs account for 13.1% of the variance of t-25OHD levels. More than half of the genetic effect on t-25OHD levels was explained by two polymorphisms (rs7935125 in NADSYN1 and rs2762941 in CYP24A1), which had not previously been investigated with respect to vitamin D metabolism. No SNPs exhibited association with f-25OHD levels. Unexpectedly, SNPs that showed univariate associations with vitamin D binding protein (DBP) levels were not associated with f-25OHD levels questioning the biological significance of these polymorphisms. The present study shows that t-25OHD levels are significantly influenced by genetic factors, however, the clinical significance of this observation remains to be defined, as variation in f-25OHD levels are marginally explained by genetic effects.