Abstract Background: The genetic etiology of osteosarcoma remains poorly understood despite the publication of a genome-wide association study. Association between HLA genetic variants and risk of several cancers has been observed, but HLA variation is not well captured by standard SNP arrays. Methods: We genotyped 207 Californian pediatric osteosarcoma cases and 696 controls of European ancestry using a custom genome-wide array supplemented with approximately 6,000 additional probes across the MHC region. We subsequently imputed 4-digit classical HLA alleles using a reference panel of 5,225 individuals who underwent high-resolution HLA typing via next-generation sequencing. Case–control comparisons were adjusted for ancestry-informative principal components, and top associations from the discovery analysis underwent replication in an independent dataset of 657 cases and 1,183 controls. Results: Three highly correlated HLA class II variants (r2 = 0.33–0.98) were associated with osteosarcoma risk in discovery analyses, including HLA-DRB1*0301 (OR = 0.52; P = 3.2 × 10−3), HLA-DQA1*0501 (OR = 0.74; P = 0.031), and HLA-DQB1*0201 (OR = 0.51; P = 2.7 × 10−3). Similar associations were observed in the replication data (Prange = 0.011–0.037). Meta-analysis of the two datasets identified HLA-DRB1*0301 as the most significantly associated variant (ORmeta = 0.62; Pmeta = 1.5 × 10−4), reaching Bonferroni-corrected statistical significance. The meta-analysis also revealed a second significant independent signal at HLA-DQA1*01:01 (ORmeta = 1.33, Pmeta = 1.2 × 10−3), and a third suggestive association at HLA-DQB1*0302 (ORmeta = 0.73, Pmeta = 6.4 × 10−3). Conclusions: Multiple independent HLA class II alleles may influence osteosarcoma risk. Impact: Additional work is needed to extend our observations to other patient populations and to clarify the potential causal mechanisms underlying these associations. Understanding immunologic contributions to the etiology of osteosarcoma may inform rational therapeutic targets. Cancer Epidemiol Biomarkers Prev; 27(10); 1151–8. ©2018 AACR.