Amplified fragment length polymorphism (AFLP) and microsatellite (SSR) markers were applied to a segregation population of 111 genotypes derived from a pseudo-testcross of hop ( Humulus lupulus L.) in order to detect quantitative trait loci (QTLs) for alpha-acid content and yield traits. A total of 199 markers (150 AFLPs, 43 SSRs, one hypothetical sex marker, five chs genes) were located on the 20 linkage groups (LGs) of the maternal and paternal maps, covering 706 and 616 cM, respectively. Due to the presence of 16 common biparental SSR markers, homology of seven LGs between parental maps could be inferred. The progeny segregated quantitatively for alpha-acid content and yield determined in the years from 2002–2006. A total of 13 putative QTLs for alpha acid content, 13 for dry cone weight and 18 for harvest index were identified on the two maps across years. Possible homologies between the detected QTLs on the two maps as well as in different years were established for all three traits. The most promising QTL for alpha acid content was identified on LG03 flanked by two AFLP markers (E-ACC-M-CAA103F*/P-ACA-M-CAC412F). From 13.80 to 36.64% higher content of alpha acids than the averages obtained in different years was observed in plants having both flanking markers. The candidate region for further characterization of QTLs for yield traits was located on LG01 where the putative QTLs for harvest index were detected on both maps in each of the 5 years. The QTLs identified represent an important improvement in alpha acids MAS and the first step towards marker-assisted breeding for hop yield.