The objectives of this study were to assess (i) the nature and frequency of polymorphism of the DQA2 gene in Chios dairy sheep and (ii) if it is associated with footrot prevalence in the population. A total of 20 DQA2 alleles/haplotypes and 78 genotypes were detected. Allele 0301was the most common, followed by haplotype 0603–1101, alleles 0501 and 0602. Furthermore, it was found that a single copy of 1101 was associated with 9.0% higher susceptibility to footrot. Regarding genotypes, 0301/0301 was the most common (12.0%), followed by 0301/0603–1101 (7.0%) and the overall effect of genotype on prevalence of footrot was statistically significant. Moreover, remarkable differences were observed, regarding frequencies of alleles/haplotypes and genotypes in Chios sheep population, when comparisons were made with breeds utilized for meat and wool production. A high diversity at the ovine DQA2 gene locus was revealed in the Chios dairy breed, suggesting that genetic selection against allele 1101 may be successful in rendering the breed less susceptible to footrot.