Evidence is increasing for a role of polymorphisms in maternal or fetal innate immune response genes in preterm birth. Toll-like receptors (TLRs) are important receptors in the innate immunity. The genotype distribution of two TLR2 single nucleotide polymorphisms (SNPs) and one TLR4 SNP were determined among 524 neonates and associated with gestational age (GA). Genomic DNA was isolated from prospectively collected blood samples and polymorphisms in TLR2 (T-16934A, RS4696480 and Arg753Gln, RS5743708) and TLR4 (Thr399Ile, RS4986791) were determined using sequence specific primers by PCR. Allele frequencies of two TLR2 SNPs and one TLR4 SNP were analyzed according to prematurity. Analysis among 305 infants, after exclusion of infants born after multiple pregnancy or because of preeclampsia, revealed significantly shorter GAs for infants carrying two polymorphic TLR2 alleles (-16934TA/AA and 753ArgGln/GlnGln) compared with infants carrying one polymorphic and one wild-type allele or two wild-type alleles (median GA 30.6 wk versus 34.1-36.8 wk, respectively, p < 0.02). Carriage of two variant TLR2 alleles potentially leads to aberrant innate immune responses, which may have contributed to very preterm birth.