In the present study, a range of benzoxazinoid compounds and phenolic acids, all known to be allelochemicals of rye, were identified and quantified in 13 rye cultivars grown at three different localities. Plant samples were collected in the spring at the time when an autumn-sown rye cover crop would be incorporated into the soil. Significant variations in content among shoots and roots were seen for all of the secondary metabolites, with non-methoxy-substituted benzoxazinoids (BX) dominating the shoots, whereas comparable levels were found in the concentrations of BX and methoxy-substituted benzoxazinoids (MBX) in the roots. This distribution of compounds may indicate different biosynthetic pathways and/or different mechanisms of action of these compounds. Concentrations not only depended on plant part, but also on the geographical location – with differences in contents of up to a factor of 5. These differences can probably be attributed to differences in growing conditions. The variation among cultivars was similar to that among geographical localities, with differences within localities of up to a factor of 7 in the shoots and a factor of 14 in the roots. In roots, the contents of the four phenolic acids and the benzoxazinoid 6-methoxybenzoxazolin-2-one (MBOA) were correlated. In shoots, the contents of the two benzoic acids were correlated with each other, whereas the two cinnamic acids were correlated with MBOA and several other benzoxazinoids. The lack of correlation between MBOA and all other benzoxazinoids in the roots of rye might indicate that a hitherto unknown synthetic pathway exists for MBOA. The genes responsible for the synthesis of some of the benzoxazinoids have never been identified, and further gene expression studies are required to assess the observed correlation between the concentration of these compounds and other benzoxazinoids for which the responsible genes are known. The present study revealed a potential for breeding rye cultivars with a high content of biologically active secondary metabolites. However, growing conditions significantly influenced the level of these compounds.