In this study, we refine a quantitative trait locus for equine osteochondrosis (OC) on horse chromosome (ECA) 2 to a genome-wide significant interval at 20.08–30.94 Mb. The marker set contained 27 newly developed microsatellites equidistantly distributed over ECA2 and 44 nucleotide polymorphisms, located in 16 positional candidate genes for OC. Genotyping was performed in 211 Hanoverian horses from 14 paternal half-sib groups. A NCDN-associated SNP and haplotype were significantly associated with OC in fetlock and/or hock joints. This study is a further step towards the identification of genes responsible for OC in horses.