Objective: A major barrier to genetic studies of osteoarthritis (OA) is the need to obtain large numbers of individuals with standardized radiographic evaluations for OA. To address this gap, we performed a genome-wide association study (GWAS) of radiographically defined tibiofemoral knee OA in 3,898 cases and 3,168 controls from 4 well-characterized North American cohorts, and we performed replication analysis of previously reported OA loci. Methods: We performed meta-analysis using a 2-stage design. Stage 1 (discovery) consisted of a GWAS meta-analysis of radiographic knee OA carried out in the Osteoarthritis Initiative and the Johnston County Osteoarthritis Project. Knee OA was defined as definite osteophytes and possible joint space narrowing or total joint replacement in one or both knees. Stage 2 (validation) was performed in the Multicenter Osteoarthritis Study and the Genetics of Osteoarthritis study. We genotyped lead meta-analysis variants (P ≤ 1 × 10−4) from stage 1 and tested the association between these variants and knee OA. We then combined results from all cohorts in a meta-analysis. Results: Lead variants from stage 1, representing 49 unique loci, were analyzed in stage 2; none met genome-wide significance in the combined analysis of stage 1 and stage 2. We validated 1 locus (rs4867568 near LSP1P3) with nominal significance (P < 0.05), which was also our top finding in the combined meta-analysis (odds ratio [OR] 0.84 [95% confidence interval (95% CI) 0.79–0.91], P = 3.02 × 10−6). We observed nominally significant associations (P < 0.05) with 3 previously reported OA loci: rs143383 in GDF5 (OR 1.12 [95% CI 1.04–1.21], P = 2.13 × 10−3), rs835487 in CHST11 (OR 0.93 [95% CI 0.85–0.99], P = 0.03), and rs8044769 in FTO (OR 1.10 [95% CI 1.03–1.19], P = 6.13 × 10−3). Conclusion: These findings provide suggestive evidence of a novel knee OA locus and confirm previously reported associations in GDF5, CHST11, and FTO.