Osteoarthritis (OA), the most prevalent form of arthritis in the elderly, is characterized by the degradation of articular cartilage and has a strong genetic component. Our aim was to identify genetic variants involved in risk of knee OA in women. A pooled genome-wide association scan with the Illumina550 Duo array was performed in 255 controls and 387 cases. Twenty-eight variants with p <1 × 10−5 were estimated to have probabilities of being false positives ≤0.5 and were genotyped individually in the original samples and in replication cohorts from the UK and the U.S. (599 and 272 cases, 1530 and 258 controls, respectively). The top seven associations were subsequently tested in samples from the Netherlands (306 cases and 584 controls). rs4140564 on chromosome 1 mapping 5′ to both the PTGS2 and PLA2G4A genes was associated with risk of knee OA in all the cohorts studied (overall odds ratio ORmh = 1.55 95% C.I. 1.30–1.85, p <6.9 × 10−7). Differential allelic expression analysis of PTGS2 with mRNA extracted from the cartilage of joint-replacement surgery OA patients revealed a significant difference in allelic expression (p <1.0 × 10−6). These results suggest the existence of cis-acting regulatory polymorphisms that are in, or near to, PTGS2 and in modest linkage disequilibrium with rs4140564. Our results and previous studies on the role of the cyclooxygenase 2 enzyme encoded by PTGS2 underscore the importance of this signaling pathway in the pathogenesis of knee OA.