Hydroxamates (HX) are major secondary metabolites synthesized by rye and are responsible for some of the unique properties of this cereal, including good tolerance of biotic and abiotic stresses and allelopathy. Recently, five genes encoding enzymes taking part in HX biosynthesis have been sequenced and characterized, which was the starting point to undertake the present study. Association analysis of the content of six HX–HBOA (2-hydroxy-1,4-benzoxazin-3-one), GDIBOA (2,4-dihydroxy-1,4-benzoxazin-3(4H)-one glucoside), DIBOA (2,4-dihydroxy-1,4-benzoxazin-3(4H)-one), GDIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3(4H)-one glucoside), DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3(4H)-one) and MBOA (6-methoxy-benzoxazolin-2(3H)-one) in the above-ground parts of plants and roots was performed on a population consisting of 102 and 121 diverse inbred lines, in 2013 and 2014, respectively. Altogether, 48 single nucleotide polymorphisms (SNPs) were found to be associated with the content of at least one HX: 20 SNPs were associated with HX synthesized in the above-ground parts of rye plants (AG-SNP), and 28 were associated with HX synthesized in the roots (R-SNP). The highest number of SNPs was present in genes ScBx1 (9) and ScBx5 (11). The majority of SNPs were affected by environmental factors, except for two: ScBx4_1702 associated with GDIBOA and MBOA contents, and ScBx5_1105 associated with HBOA content in roots.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-016-0356-3) contains supplementary material, which is available to authorized users.