We present a new set of 11 polymorphic microsatellite primer sequences for use with Humulus lupulus. Microsatellite-enriched libraries for GAn and GTn types of repeats were produced. Sequencing of 72 clones revealed 42 unique inserts containing microsatellites, out of which 19 primer pairs were designed and microsatellite amplification was tested on 39 wild hops and cultivars. Eleven primer pairs showed single locus amplification with 2–13 alleles, average 7.2, of which 17 unique alleles were discovered. One primer pair amplified too strong stutter bands, one locus was monomorphic and multilocus amplification was obtained with the remaining six primer pairs.