Plant genomes are now sequenced rapidly and inexpensively. In silico approaches allow efficient development of simple sequence repeat markers, also known as microsatellite markers, from these sequences. A search of the genome sequence of 'Jefferson' hazelnut (Corylus avellana L.) identified 8,708 tri-nucleotide simple sequence repeats with at least five repeat units, and stepwise removal of the less promising sequences led to the development of 150 polymorphic markers. Fragments in the 'Jefferson' sequence containing tri-nucleotide repeats were used as references and aligned with genomic sequences from seven other cultivars. Following in silico alignment, sequences that showed variation in number of repeat units were selected and primer pairs were designed for 243 of them. Screening on agarose gels identified 173 as polymorphic. Removal of duplicate and previously published sequences reduced the number to 150, for which fluorescent primers and capillary electrophoresis were used for amplicon sizing. These were characterized using 50 diverse hazelnut accessions. Of the 150, 132 generated the expected one or two alleles per accession while 18 amplified more than two amplicons in at least one accession. Diversity parameters of the 132 marker loci averaged 4.73 for number of alleles, 0.51 for expected heterozygosity (He), 0.49 for observed heterozygosity (Ho), 0.46 for polymorphism information content (PIC), and 0.04 for frequency of null alleles. The clustering of the 50 accessions in a dendrogram constructed from the 150 markers confirmed the wide genetic diversity and presence of three of the four major geographic groups: Central European, Black Sea, and Spanish-Italian. In the mapping population, 105 loci segregated, of which 101 were assigned to a linkage group (LG), with positions well-dispersed across all 11 LGs. These new markers will be useful for cultivar fingerprinting, diversity studies, genome comparisons, mapping, and alignment of the linkage map with the genome sequence and physical map.