The objective of the paper was to identify the number of major loci explaining caffeine content in coffee seeds. Investigations were based on previously published results: (1) Caffeine binds to chlorogenic acids in a 1:1 molecular ratio; (2) Between species, the caffeine content is correlated to the chlorogenic acid content; (3) Only a part of chlorogenic acids is bound to caffeine. Especially, the content ratio between caffeine and chlorogenic acids varied between species. For identifying the number of major loci, a quantitative trait locus (QTL) approach was carried out using an interspecific cross between two highly differentiated species-Coffea liberica dewevrei and Coffea pseudozanguebariae, the latter being a caffeine-free species. As main finding, two QTLs, i.e., RCQ1 and CQA1, were identified allowing us to explain up to 97 % of the caffeine content variance. RCQ1 explained variation of the caffeine/chlorogenic acid ratio and was genetically independent of the second QTL. The latter explained the part of the caffeine content which was dependent on the chlorogenic acid content. The findings also confirmed that only a part of chlorogenic acids were trapped by caffeine, as in wild species.