Anal furunculosis (AF) is a chronic inflammatory disease of perianal tissues that particularly affects German Shepherd dogs (GSD). An immune-mediated aetiopathogenesis is suggested by T-cell infiltration, upregulated cytokine gene expression, clinical response to ciclosporin therapy and a strong genetic association with the DLA-DRB1*00101 allele. Given the close proximity of TNFA and DLA-DRB1 in the canine major histocompatibility complex (MHC), together with the strong linkage disequilibrium (LD) observed across this region, the primary disease association could be with either locus. We have investigated whether there may be an association of AF with TNFA gene polymorphism in GSDs. Cohorts of AF-affected and AF-unaffected GSDs of known dog leucocyte antigen (DLA) class II profile were genotyped for 10 single nucleotide polymorphisms (SNPs) in the canine TNFA locus using Sequenom iPLEX technology. Seven discrete TNFA haplotypes were identified in GSDs for combinations of these SNPs. TNFA haplotype frequencies were compared in cases and controls. The TNFA haplotype 3 (ATCGTTACGG), was at significantly increased frequency in cases (29% vs 15%, OR 2.5, 95% CI 1.4-4.8; P = 0.003). All seven discrete TNFA SNP haplotypes were examined for their association with DLA-DRB1/DQA1/DQB1 established haplotypes. TNFA haplotype 3 was preferentially associated with both DLA-DRB1*00101(3A)- and DLA-DRB1*00102(3B)-positive haplotypes. The DLA-DRB1* 00101/TNFA-3A haplotype was significantly associated with AF (19.3% vs 5.8%; OR 3.7, 95% CI: 1.5-8.9; P = 0.003), whereas the DLA-DRB1*00102/TNFA-3B haplotype was not (P = NS). These findings suggest that susceptibility to AF in GSDs is primarily associated with DLA-DRB1*00101 and any association with the TNFA locus is secondary and is likely to be because of LD.