The polymerase chain reaction (PCR) has become a standard procedure in plant genetics, and is the basis for many emerging genomics approaches to mapping and gene identification. One advantage of PCR is that sequence information for primer sets can be exchanged between laboratories, obviating the need for exchange and maintenance of biological materials. Repeatability of primer sets, whereby the same products are amplified in different laboratories using the same primer set, is important to successful exchange and utilization. We have developed several hundred sequence-tagged site (STS) primer sets for wheat and barley. The ability of the primer sets to generate reproducible amplifications in other laboratories has been variable. We wished to empirically determine the properties of the primer sets that most influenced repeatability. A total of 96 primer sets were tested with four genomic DNA samples on each of four thermocyclers. All major bands were repeatable across all four thermocylers for approximately 50% of the primer sets. Characteristics most often associated with differences in repeatability included primer GC content and 3'-end stability of the primers. The propensity for primer-dimer formation was not a factor in repeatability. Our results provide empirical direction for the development of repeatable primer sets.