•HLA typing may help to identify patients with ASSD.•HLA-DRB1*03:01 and HLA-B1*08:01 are predisposition markers of ASSD.•HLA-DRB1*07:01 is a protective allele against ASSD.•HLA-DRB1*03:01 increases the risk of developing anti-Jo-1 autoantibodies in ASSD.•Smoking does not seem to influence anti-Jo-1 antibody positivity in ASSD. To investigate the human leukocyte antigen (HLA) association with anti-synthetase syndrome (ASSD). We conducted the largest immunogenetic HLA-DRB1 and HLA-B study to date in a homogeneous cohort of 168 Caucasian patients with ASSD and 486 ethnically matched healthy controls by sequencing-based-typing. A statistically significant increase of HLA-DRB1*03:01 and HLA-B*08:01 alleles in patients with ASSD compared to healthy controls was disclosed (26.2% versus 12.2%, P=1.56E–09, odds ratio–OR 95% confidence interval–CI=2.54 1.84–3.50 and 21.4% versus 5.5%, P=18.95E–18, OR 95% CI=4.73 3.18–7.05; respectively). Additionally, HLA-DRB1*07:01 allele was significantly decreased in patients with ASSD compared to controls (9.2% versus 17.5%, P=0.0003, OR 95% CI=0.48 0.31–0.72). Moreover, a statistically significant increase of HLA-DRB1*03:01 allele in anti-Jo-1 positive compared to anti-Jo-1 negative patients with ASSD was observed (31.8% versus 15.5%, P=0.001, OR 95% CI=2.54 1.39–4.81). Similar findings were observed when HLA carrier frequencies were assessed. The HLA-DRB1*03:01 association with anti-Jo-1 was unrelated to smoking history. No HLA differences in patients with ASSD stratified according to the presence/absence of the most representative non-anti-Jo-1 anti-synthetase autoantibodies (anti-PL-12 and anti-PL-7), arthritis, myositis or interstitial lung disease were observed. Our results support the association of the HLA complex with the susceptibility to ASSD.