Cultivar identification is important to the barley malting and brewing industries. As breeders work within narrow gene pools, the more traditional methods of identification, such as seed morphology and protein electrophoresis, have become ineffective. Modern tools of biotechnology that can detect DNA sequence differences have been employed to identify cultivars from a narrow genetic base. Of the techniques, the polymerse chain reaction with short, random primers (PCR-RAPD) was effective in distinguishing among a set of six-rowed malting barley cultivars. However, the reported lack reproducibility of the PCR-RAPD technique among laboratories has limited its widespread acceptance as a detection tool. To address this problem, we cloned and sequenced RAPD fragments that mapped to polymorphic regions of six-rowed malting barley. Several single nucleotide polymorphisms (SNPs) were identified between Robust and Stander, a key comparison. The DNA sequence information was used to design a sequence-tagged–site PCR primer pair that amplified an 800 base pair DNA fragment. This fragment, when digested with Alu1, resulted in a pattern that could distinguish Stander from Robust and vice versa. Another procedure, amplified fragment length polymorphism (AFLP), was investigated as to its ability to reliably differentiate among certain six-rowed malting barley cultivars. Numerous AFLPs were found among the cultivars, and these will be reported. This work has identified alternative procedures that can distinguish certain six-rowed malting barley cultivars in addition to the previously reported PCR-RAPD method.