The butyrophilin-like protein 2 gene (BTNL2) within the class III region of the major histocompatibility complex genomic region was identified as a rheumatoid arthritis (RA) susceptibility gene by exome sequencing (19 RA cases) with stepwise filtering analysis, and then validated by Sanger sequencing and association analysis using 432 cases and 432 controls. Logistic regression of the Sanger-sequenced single-nucleotide variants in an association study of 432 cases and 432 controls showed that 12 non-synonymous single-nucleotide polymorphisms (SNPs) in BTNL2 were significantly associated with RA. The lowest P-values were obtained from three SNPs, rs41521946, rs28362677 and rs28362678, which were in absolute linkage disequilibrium: P=4.55E-09, odds ratio=1.88, 95% confidence interval=1.52-2.33. The BTNL2 locates on chromosome 6 between HLA-DRB1 and NOTCH4, and is 170 kb apart from these two genes. Although DRB1 and NOTCH4 were reported to be RA-susceptible, the three BTNL2 SNPs retained significant association with RA when evaluated by the logistic regression with the adjustment for RA-susceptible HLA-DRB1 alleles in Japanese or rs2071282-T in NOTCH4: P=0.0156 and P=0.00368, respectively. These results suggest that the three non-synonymous SNPs in BTNL2 confer RA risk independently from HLA-DRB1 and NOTCH4.