The development of DNA technology has provided a number of methods, which eliminate any influence of environmental factors, to detect differences at the DNA sequence level for evaluation of hop genotypes. Most of these methods in hops used unknown DNA sequences (e.g., AFLP) or specific DNA sequences (e.g., SSR) in non-coding regions of the genome. A new trend in molecular methods is to develop new molecular markers in specific sequences, near or within structural genes. Recently, markers in intron, 5' and 3'UTR were identified in several hop genes. Increasing sequence information of hop genes in Genebank provides additional opportunities to develop new gene specific molecular markers. In this study, we report new efficient and reliable expressed sequence tag microsatellite repeat (EST-SSR) molecular markers. We used forty primer pairs for twenty-nine gene specific sequences. We evaluated them for identification and clustering in eight hops cultivars. Gene-specific based clustering was compared to clustering based on SSR, AFLP markers and on a combination of molecular markers. These molecular markers are ideal for DNA fingerprinting, for control of authenticity and purity of varieties, for evaluation of genetic diversity in genetic resources, etc. They are readily applicable in breeding, and for genetic linkage mapping and quantitative trait loci (QTL) analysis.