Background and aims: Patients with type 1 diabetes have antibodies to ZnT8 protein is encoded by SLC30A8. The C-allele of the R325 W variant in SLC30A8 is associated with type 2 diabetes and reduced beta-cell function in non-diabetic subjects. Our aim was to assess the prevalence of ZnT8 autoantibodies (ZnT8A) in patients with adult-onset diabetes, and to characterize associations between ZnT8A and phenotype, as well as SLC30A8 and HLA-DQB1 genotypes. Methods: ZnT8A were analyzed in patients diagnosed with diabetes >35 years (type 1 diabetes: n = 274; Latent autoimmune diabetes in adults (LADA): n = 294). SLC30A8 R325 W (rs13266634) and HLA-DQB1 alleles were genotyped in all patients and 537 non-diabetic control subjects. Results: ZnT8A were significantly more prevalent in LADA (34.3%) compared to adult-onset type 1 diabetes (18.7%, p < 0.0001). Among the patients with adult-onset type 1 diabetes, ZnT8A were associated with shorter disease duration [4.4 (6.0) vs. 10.8 (11.2) years, p < 0.0001], whereas no such association was observed among patients with LADA. The SLC30A8 R325 W variant was associated with LADA with low GADA levels [SLC30A8 CC: OR (95% CI): 1.46 (1.00-2.13), p = 0.049], and reduced insulin secretion among the non-diabetic subjects and the patients with LADA. Conclusion: ZnT8A were more common and more persistent in patients with LADA compared to adult-onset type 1 diabetes, but their presence was not associated with specific phenotypic characteristics. The SLC30A8 CC genotype adds to the genetic heterogeneity of LADA linked to GADA reactivity.