Based on the recent observation that Toll-like receptors (TLRs) may be involved in the pathogenesis of osteoarthritis (OA) we explored the possibility that human TLR gene polymorphisms are associated with OA. Two separate populations were studied in a two-stage case-control study with a total of 503 OA patients and 428 healthy controls. The TLR-2, TLR-4, and TLR-9 genotypes were assessed by real-time polymerase chain reaction. Our data demonstrated a lack of association among TLR-2, TLR-4, and TLR-9 (T-1237C) polymorphisms and the risk of developing OA in both stages of the study. T-1486C was significantly associated with OA in both populations with G1635A of TLR-9 gene was found to be significantly associated with OA when the two populations were combined. Stratifying the samples by K-L score there were significant differences in the genotype of the TLR-9 T-1486C and G1635A between OA of the knee grade 4 and controls. In haplotype analyses, the haplotype TTG and TTA revealed higher risk of OA and TCA confers a lower risk of OA in combined population. The present results demonstrate that TLR-9 polymorphisms, in particular T-1486C is significantly associated with OA. TLR-9 gene polymorphisms may play a role in the etiology of knee OA.